Abstract
Reproduction induces increased food intake across females of many animal species1,2,3,4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop—a stomach-like organ—after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Source data are provided with this paper.
Change history
11 December 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-3013-2
References
Carvalho, G. B., Kapahi, P., Anderson, D. J. & Benzer, S. Allocrine modulation of feeding behavior by the Sex Peptide of Drosophila. Curr. Biol. 16, 692–696 (2006).
Gittleman, J. L. & Thompson, S. D. Energy allocation in mammalian reproduction. Am. Zool. 28, 863–875 (1988).
Johnson, M. L., Saffrey, M. J. & Taylor, V. J. Gastrointestinal capacity, gut hormones and appetite change during rat pregnancy and lactation. Reproduction 157, 431–443 (2019).
Speakman, J. R. The physiological costs of reproduction in small mammals. Phil. Trans. R. Soc. Lond. B 363, 375–398 (2008).
Dey, S. et al. Cyclic regulation of sensory perception by a female hormone alters behavior. Cell 161, 1334–1344 (2015).
Grunwald Kadow, I. C. State-dependent plasticity of innate behavior in fruit flies. Curr. Opin. Neurobiol. 54, 60–65 (2019).
Krashes, M. J. et al. A neural circuit mechanism integrating motivational state with memory expression in Drosophila. Cell 139, 416–427 (2009).
Bai, L. et al. Genetic identification of vagal sensory neurons that control feeding. Cell 179, 1129–1143 e.23 (2019).
Han, W. et al. A neural circuit for gut-induced reward. Cell 175, 887–888 (2018).
Muller, P. A. et al. Microbiota modulate sympathetic neurons via a gut-brain circuit. Nature 583, 441–446 (2020).
Talbot, J. et al. Feeding-dependent VIP neuron–ILC3 circuit regulates the intestinal barrier. Nature 579, 575–580 (2020).
Tan, H.-E. et al. The gut–brain axis mediates sugar preference. Nature 580, 511–516 (2020).
Zimmerman, C. A. et al. A gut-to-brain signal of fluid osmolarity controls thirst satiation. Nature 568, 98–102 (2019).
Kim, D.-Y. et al. A neural circuit mechanism for mechanosensory feedback control of ingestion. Nature 580, 376–380 (2020).
Augustine, V. et al. Temporally and spatially distinct thirst satiation signals. Neuron 103, 242–249.e4 (2019).
Drokhlyansky, E. et al. The human and mouse enteric nervous system at single-cell resolution. Cell 182, 1606–1622.e23 (2020).
Kaelberer, M. M. et al. A gut–brain neural circuit for nutrient sensory transduction. Science 361, eaat5236 (2018).
Lasrado, R. et al. Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science 356, 722–726 (2017).
Williams, E. K. et al. Sensory neurons that detect stretch and nutrients in the digestive system. Cell 166, 209–221 (2016).
Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 e.22 (2018).
Miguel-Aliaga, I., Jasper, H. & Lemaitre, B. Anatomy and physiology of the digestive tract of Drosophila melanogaster. Genetics 210, 357–396 (2018).
Stoffolano, J. G. Jr & Haselton, A. T. The adult Dipteran crop: a unique and overlooked organ. Annu. Rev. Entomol. 58, 205–225 (2013).
Cao, C. & Brown, M. R. Localization of an insulin-like peptide in brains of two flies. Cell Tissue Res. 304, 317–321 (2001).
Cognigni, P., Bailey, A. P. & Miguel-Aliaga, I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab. 13, 92–104 (2011).
Rulifson, E. J., Kim, S. K. & Nusse, R. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296, 1118–1120 (2002).
Dus, M. et al. Nutrient sensor in the brain directs the action of the brain–gut axis in Drosophila. Neuron 87, 139–151 (2015).
Kim, S. K. & Rulifson, E. J. Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells. Nature 431, 316–320 (2004).
Lee, G. & Park, J. H. Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167, 311–323 (2004).
Edgecomb, R. S., Harth, C. E. & Schneiderman, A. M. Regulation of feeding behavior in adult Drosophila melanogaster varies with feeding regime and nutritional state. J. Exp. Biol. 197, 215–235 (1994).
McCormick, J. & Nichols, R. Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster. J. Comp. Neurol. 338, 279–288 (1993).
Richer, S., Stoffolano, J. G. Jr, Yin, C. M. & Nichols, R. Innervation of dromyosuppressin (DMS) immunoreactive processes and effect of DMS and benzethonium chloride on the Phormia regina (Meigen) crop. J. Comp. Neurol. 421, 136–142 (2000).
Holman, G. M., Cook, B. J. & Nachman, R. J. Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut. Comp. Biochem. Physiol. C 85, 329–333 (1986).
Egerod, K. et al. Molecular cloning and functional expression of the first two specific insect myosuppressin receptors. Proc. Natl Acad. Sci. USA 100, 9808–9813 (2003).
Harshman, L. G., Loeb, A. M. & Johnson, B. A. Ecdysteroid titers in mated and unmated Drosophila melanogaster females. J. Insect Physiol. 45, 571–577 (1999).
Schwedes, C. C. & Carney, G. E. Ecdysone signaling in adult Drosophila melanogaster. J. Insect Physiol. 58, 293–302 (2012).
Reiff, T. et al. Endocrine remodelling of the adult intestine sustains reproduction in Drosophila. eLife 4, e06930 (2015).
Scopelliti, A. et al. A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila. Cell Metab. 29, 269–284.e10 (2019).
Davey, K. G. & Treherne, J. E. Studies on crop function in the cockroach (Periplaneta americana L.). J. Exp. Biol. 41, 513–524 (1964).
Itskov, P. M. et al. Automated monitoring and quantitative analysis of feeding behaviour in Drosophila. Nat. Commun. 5, 4560 (2014).
Chapman, T. & Partridge, L. Female fitness in Drosophila melanogaster: an interaction between the effect of nutrition and of encounter rate with males. Proc. R. Soc. Lond. B 263, 755–759 (1996).
Poels, J. et al. Myoinhibiting peptides are the ancestral ligands of the promiscuous Drosophila sex peptide receptor. Cell. Mol. Life Sci. 67, 3511–3522 (2010).
Min, S. et al. Control of feeding by piezo-mediated gut mechanosensation in Drosophila. Preprint at https://www.biorxiv.org/content/10.1101/2020.09.11.293712v1.abstract (2020).
Wang, P., Jia, Y., Liu, T., Jan, Y-N. & Zhang, W. Visceral mechano-sensing neurons control Drosophila feeding by using piezo as a sensor. Neuron https://doi.org/10.1016/j.neuron.2020.08.017 (2020).
Sieber, M. H. & Spradling, A. C. Steroid signaling establishes a female metabolic state and regulates SREBP to control oocyte lipid accumulation. Curr. Biol. 25, 993–1004 (2015).
Brunton, P. J. & Russell, J. A. The expectant brain: adapting for motherhood. Nat. Rev. Neurosci. 9, 11–25 (2008).
Hoekzema, E. et al. Pregnancy leads to long-lasting changes in human brain structure. Nat. Neurosci. 20, 287–296 (2017).
Ameku, T., Beckwith, H., Blackie, L. & Miguel-Aliaga, I. Food, microbes, sex and old age: on the plasticity of gastrointestinal innervation. Curr. Opin. Neurobiol. 62, 83–91 (2020).
Hadjieconomou, D. et al. Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat. Methods 8, 260–266 (2011).
Burke, C. J. & Waddell, S. Remembering nutrient quality of sugar in Drosophila. Curr. Biol. 21, 746–750 (2011).
Fujita, M. & Tanimura, T. Drosophila evaluates and learns the nutritional value of sugars. Curr. Biol. 21, 751–755 (2011).
Ribeiro, C. & Dickson, B. J. Sex peptide receptor and neuronal TOR/S6K signaling modulate nutrient balancing in Drosophila. Curr. Biol. 20, 1000–1005 (2010).
Ikeya, T., Galic, M., Belawat, P., Nairz, K. & Hafen, E. Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12, 1293–1300 (2002).
Miyamoto, T., Slone, J., Song, X. & Amrein, H. A fructose receptor functions as a nutrient sensor in the Drosophila brain. Cell 151, 1113–1125 (2012).
Jenett, A. et al. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep. 2, 991–1001 (2012).
Asahina, K. & Anderson, D. Neuropeptide-GAL4 constructs and insertions. Reference report FBrf0222772 (FlyBase, 2013).
Tracey, W. D. Jr, Wilson, R. I., Laurent, G. & Benzer, S. painless, a Drosophila gene essential for nociception. Cell 113, 261–273 (2003).
Thorne, N. & Amrein, H. Atypical expression of Drosophila gustatory receptor genes in sensory and central neurons. J. Comp. Neurol. 506, 548–568 (2008).
de Navas, L., Foronda, D., Suzanne, M. & Sánchez-Herrero, E. A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila. Mech. Dev. 123, 860–867 (2006).
Hudry, B., Viala, S., Graba, Y. & Merabet, S. Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay. BMC Biol. 9, 5 (2011).
Park, D., Veenstra, J. A., Park, J. H. & Taghert, P. H. Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS ONE 3, e1896 (2008).
Zhan, Y. P., Liu, L. & Zhu, Y. Taotie neurons regulate appetite in Drosophila. Nat. Commun. 7, 13633 (2016).
Guo, Z., Driver, I. & Ohlstein, B. Injury-induced BMP signaling negatively regulates Drosophila midgut homeostasis. J. Cell Biol. 201, 945–961 (2013).
Diao, F., Elliott, A. D., Diao, F., Shah, S. & White, B. H. Neuromodulatory connectivity defines the structure of a behavioral neural network. eLife 6, e29797 (2017).
Balakireva, M., Gendre, N., Stocker, R. F. & Ferveur, J. F. The genetic variant Voila causes gustatory defects during Drosophila development. J. Neurosci. 20, 3425–3433 (2000).
Song, W., Veenstra, J. A. & Perrimon, N. Control of lipid metabolism by tachykinin in Drosophila. Cell Rep. 9, 40–47 (2014).
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. & Davis, R. L. Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765–1768 (2003).
Hudry, B., Khadayate, S. & Miguel-Aliaga, I. The sexual identity of adult intestinal stem cells controls organ size and plasticity. Nature 530, 344–348 (2016).
Nicolaï, L. J. et al. Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila. Proc. Natl Acad. Sci. USA 107, 20553–20558 (2010).
Sugimura, K. et al. Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons. J. Neurosci. 23, 3752–3760 (2003).
Hamada, F. N. et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature 454, 217–220 (2008).
Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T. & Bate, M. Altered electrical properties in Drosophila neurons developing without synaptic transmission. J. Neurosci. 21, 1523–1531 (2001).
Barolo, S. et al. A notch-independent activity of suppressor of Hairless is required for normal mechanoreceptor physiology. Cell 103, 957–970 (2000).
Masuyama, K., Zhang, Y., Rao, Y. & Wang, J. W. Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet. 26, 89–102 (2012).
Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Sarov, M. et al. A genome-wide resource for the analysis of protein localisation in Drosophila. eLife 5, e12068 (2016).
Baena-Lopez, L. A., Alexandre, C., Mitchell, A., Pasakarnis, L. & Vincent, J. P. Accelerated homologous recombination and subsequent genome modification in Drosophila. Development 140, 4818–4825 (2013).
Diao, F. et al. Plug-and-play genetic access to Drosophila cell types using exchangeable exon cassettes. Cell Rep. 10, 1410–1421 (2015).
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).
Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Peabody, N. C. et al. Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death. J. Neurosci. 28, 14379–14391 (2008).
Géminard, C., Rulifson, E. J. & Léopold, P. Remote control of insulin secretion by fat cells in Drosophila. Cell Metab. 10, 199–207 (2009).
Schoofs, L. et al. Isolation, identification, and synthesis of PDVDHFLRFamide (SchistoFLRFamide) in Locusta migratoria and its association with the male accessory glands, the salivary glands, the heart, and the oviduct. Peptides 14, 409–421 (1993).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Adams, D. C. & Otárola-Castillo, E. geomorph: an R package for the collection and analysis of geometric morphometric shape data. Methods Ecol. Evol. 4, 393–399 (2013).
Koyama, L. A. J. et al. Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila. PLoS Biol. 18, e3000567 (2020).
Lopes, G. et al. Bonsai: an event-based framework for processing and controlling data streams. Front. Neuroinform. 9, 7 (2015).
Rodriguez, A., Ehlenberger, D. B., Dickstein, D. L., Hof, P. R. & Wearne, S. L. Automated three-dimensional detection and shape classification of dendritic spines from fluorescence microscopy images. PLoS ONE 3, e1997 (2008).
Séjourné, J. et al. Mushroom body efferent neurons responsible for aversive olfactory memory retrieval in Drosophila. Nat. Neurosci. 14, 903–910 (2011).
Plaçais, P. Y. et al. Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila. Nat. Neurosci. 15, 592–599 (2012).
Buchon, N. et al. Morphological and molecular characterization of adult midgut compartmentalization in Drosophila. Cell Rep. 3, 1725–1738 (2013).
Leader, D. P., Krause, S. A., Pandit, A., Davies, S. A. & Dow, J. A. T. FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data. Nucleic Acids Res. 46, D809–D815 (2018).
Kohl, J. & Dulac, C. Neural control of parental behaviors. Curr. Opin. Neurobiol. 49, 116–122 (2018).
Ahmed, S. M. H. et al. Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila. Nature 584, 415–419 (2020).
Zipper, L., Jassmann, D., Burgmer, S., Görlich, B. & Reiff, T. Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARγ- homolog Eip75B in Drosophila. eLife. 9, e55795 (2020).
Acknowledgements
We thank C. Alexandre, J. Cordero, A. Gould, B. Hudry, M. Landgraf, P. Léopold, O. Riabinina, I. Salecker, L. Schoofs, J. Simpson and Y. Zhu for providing reagents. S. Austin and M. Hartl contributed to the early characterization of crop innervation. C. Alexandre provided assistance with the design of the Ms mutant. We thank J. Jacobson and T. Lopes for supporting our work experimentally, and G. Salbreux for initial advice on modelling, respectively. We thank L. O’Brien and L. A. J. Koyama for providing advice on Bellymount. C. Whilding assisted with imaging quantifications. V. Papayannopoulos provided comments on the manuscript. This work was funded by an ERC Advanced Grant and a BBSRC grant to I.M.-A. (ERCAdG 787470 ‘IntraGutSex’ and BB/N000528/1, respectively), and MRC intramural funding to I.M.-A.
Author information
Authors and Affiliations
Contributions
D.H. and I.M.-A. designed and conceived the study. D.H. and G.K. performed most of the experiments and analysed data. P.G. conducted crop enlargement, feeding and fecundity experiments, developed ways to quantify crop enlargement and analysed data. A.M. conducted some of the immunohistochemistry and fecundity experiments. L.B. conducted immunohistochemistry experiments and acquired and analysed feeding and crop enlargement videos. T.A. conducted some immunohistochemistry and RT–qPCR experiments. C.S. assisted with fecundity experiments, fly husbandry and video recordings. A.d M. performed phylogenetic analyses. F.D. and B.H.W. contributed the MsTGEM-Gal4 mutant/driver line. A.E.X.B. provided the mathematical model. P.-Y.P. and T.P. hosted and trained D.H. to perform in vivo brain calcium imaging experiments, P.-Y.P performed calcium imaging experiments and analysed these data. I.M-A. wrote the manuscript, with contributions from D.H.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Innervation of the anterior portion of the adult Drosophila intestine.
a, Schematic summary of the innervation of the anterior portion of the adult fly intestine, encompassing foregut, crop and anterior midgut. b, Pan-neuronal nSyb-Gal4 driver expression visualized with EGFP (from UAS-FB1.1 reporter) in green. Gut muscles are highlighted in blue with phalloidin staining. In all subsequent panels, driver expression is in green and phalloidin staining in blue. Abbreviations are as per a. c, Cell number quantifications of the enteric nervous system (ENS) ganglia and secretory glands associated with the adult anterior midgut. d–d″, Direct innervation of the crop by neurons located in the central nervous system. d′, Projections emanating from the insulin-producing neurons in the PI (labelled with Ilp2-3-Gal4-driven expression of UAS-FB1.1-derived EGFP in green) innervate the crop and anterior midgut. Neuronal nuclei are labelled with anti-Elav antibody in red, and gut muscles are labelled in blue with phalloidin. d″, The axonal projections of these insulinergic neurons are visualized using immunostaining for Ilp2 peptide in red. e–e″, Innervation of the crop by peripheral neurons. Taste receptor-expressing neurons visualized with the Gr43aKI-Gal4 driver; gut muscles are labelled with phalloidin. The boxed area in e′ highlights the cell bodies of ENS-like sensory neurons located in the HCG. e″ shows their projections on the crop muscle lobes (arrow). In d–e″, arrowheads point to the paired nerves that innervate the crop. f–j, Spatially restricted Gal4 drivers or antibodies reveal distinct crop-innervating neuronal subsets. In all panels, Gal4 expression is visualized with EGFP (from UAS-FB1.1 reporter) in green, and gut muscles are highlighted in blue with phalloidin staining. f, Dh44-Gal4 expression. Dh44-Gal4-positive cell bodies in the PI (top dashed box) project to the HCG (bottom dashed boxed) and crop through the crop nervi. They also innervate the anterior midgut. No Dh44-Gal4-positive cell bodies are apparent in the HCG. DAPI labels the nuclei of the brain–gut axis in cyan. g, Mip-Gal4-positive cell bodies are found in both the PI and HCG (dashed boxes). They extend projections to the anterior midgut, and along the crop nervi towards the crop. h, Glucagon-like adipokinetic hormone Akh (labelled with an anti-Akh antibody in red) is produced by cell bodies located in the paired corpoca cardiaca (CC) glands and is apparent in their projections along the crop nervi up to the junction between crop duct and lobes. i, Expression of a pain-Gal4 reporter for painless (coding for a TRPA channel that detects noxious heat and mechanical stimuli) in a subset of ENS neurons in the HCG (dashed box), pointing to their possible mechanosensory identity. j, Expression of a Gr28a-Gal4 reporter for Gustatory receptor 28a in two HCG cell bodies (dashed box), suggestive of chemosensory identity. Their neurites populate the anterior midgut and their putative axons project along the recurrent nerve (RN). k, The Aug21-Gal4 reporter reveals short local projections from the corpus allatum around the foregut and anterior midgut. l, m, The use of Hox gene reporters allows labelling of large population of central neurons in thoracico-abdominal ganglion segments. No neurons in the Ubx-Gal4 (l) or abdA-Gal4 (m) expression domains contribute to the innervation of the crop of anterior midgut. Gal4 expression is visualized with EGFP (from UAS-FB1.1 reporter) in green, and gut muscles are highlighted in blue with phalloidin staining. Neuronal nuclei are visualized in red with anti-Elav (SG = salivary gland). Scale bars, 50 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions.
Extended Data Fig. 2 Intestinal transit dynamics and dietary regulation of crop enlargement.
a, Schematic summarizing ad libitum and starvation–re-feeding assays using dye-laced food. b–b‴, Transit of dye-laced food at specific time points after ingestion. b, Gut dissected 10 s after feeding initiation; food is apparent in the crop duct and begins to enter the crop. b′, Gut dissected 40 s after feeding initiation; food fills the crop duct, crop, and begins to enter the midgut. b″, Gut dissected 2 min after feeding initiation; food fills the crop, crop duct and midgut. b‴, Gut dissected 40 min after feeding initiation; food fills the crop, crop duct, midgut and has now reached the hindgut and rectal ampulla. All panels show dissected adult fly intestines, anterior (left) posterior (right). c, c′, Frequency histograms derived from in vivo food ingestion videos (see Supplementary Video 1 for a representative example) showing a larger number of flies with faster transit times of food to the crop (c) compared to midgut (c′). d, Quantification of crop area revealed that re-feeding after starvation results in larger crops than ad libitum feeding. e–e″, Representative dissected guts of a starved fly (e, 16 h starvation on 1% agar), starved–refed fly (e′, 16 h starvation on 1% agar, refed for 20 min on dye-laced standard food), ad libitum-fed fly (e″, fed on dye-laced standard food for 2 h). f, Ability of different food sources to elicit crop enlargement. These are categorized as palatable (P) and/or nutritious (N) using filled boxes if true and empty boxes if false (see Methods for further details of the different diets). In this and all subsequent ranked data panels, crop size was ranked as one of four categories: small (S), medium (M), large (L) and very large (VL). Graphs are colour-coded from light to dark shades of red corresponding to increasing size of the crop. Data are displayed as percentages. Scale bars, 500 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 3 Characterization of Ms expression.
a, Schematic depicting Ms neuronal subtypes. Dashed boxes highlight the main sites of Ms expression: around 30 neuronal cell bodies in the PI, around 5 enteric neurons located in the HCG and neuronal projections in the HCG and on the crop muscles. b, c, Single-cell Flybow clones of Ms-Gal4-expressing neurons (in red); gut muscle labelled with phalloidin (in blue). b, The PI and HCG where the Ms cell bodies reside are boxed. No Ms neurons have been labelled in the PI, but a single-cell, mCitrine-positive clone (in red) reveals an HCG neuron that innervates the crop muscle. Inset shows a single-cell clone of a second type of HCG Ms-Gal4-expressing neuron that only extends local projections. c, Single-cell FlyBow48 clone of a large PI Ms-Gal4-expressing neuron. The main projection bifurcates, with one shorter (putatively dendritic) branch projecting towards the suboesophageal zone (SEZ) (empty arrows), and a longer (axonal) branch projecting towards the midgut/crop (arrows). d, d′, Co-expression of the dendritic marker DenMark (in red) and membrane marker Venus shown (in green) from Ms-Gal4 reveals relative DenMark enrichment in their SEZ projections (d), consistent with dendritic nature. Venus enrichment is apparent in the crop nerve (d′), consistent with its axonal identity. Top left arrow points to the crop nerve, and bottom arrow points to where it terminates. e, Quantification of fluorescence for DenMark and Venus in SEZ (top) crop nerve (bottom) projections. f–j″, Ms-Gal4 expression, visualized by EGFP from the UAS-FB1.1 reporter (in green). f, Overview of Ms-Gal4-positive intestinal innervation; Ms-positive neurites are apparent on the crop, anterior midgut and posterior hindgut (rectal ampulla). Neuronal nuclei are stained with an anti-Elav antibody in red, and gut muscles are labelled in blue with phalloidin. g, Ms-Gal4 expression in heart-innervating neurons; heart muscles are labelled in blue with phalloidin. h, Ms-Gal4 expression in peripheral neurons that innervate the ovaries, oviduct and spermatheca (SP). i–i″, Co-expression of Ms-Gal4 and Ms peptide (in red) in a cluster of PI neurons; arrows and arrowheads point to big and small PI Ms neuron subtypes, respectively. i and i′ show single-channel images for Ms-Gal4 and anti-Ms antibody, respectively. The merged image is shown in i″. j–j″, Co-expression of Ms-Gal4 and Ms transcript (visualized using single-molecule RNA fluorescence in situ hybridization in red) in the same cluster of PI neurons. j and j′ show single-channel images for Ms-Gal4 and Ms transcript, respectively. The merged image is shown in i″. k–k′, Ms protein reporter expression (in green). Ms peptide is in red and gut muscles are labelled with phalloidin in blue. k, Co-expression between the Ms protein reporter Ms peptide in the nervous system, and in neuronal projections towards the gut. Ms and the Ms protein reporter are co-expressed by the PI Ms neurons (boxed and inset). k′, The Ms protein reporter also labels axonal projections innervating the crop muscles. l–q″, Expression (or lack thereof) of neuropeptides and other markers in the Ms-expressing neurons in the PI or HCG. For each letter, the first panel shows double staining, the second and third panels show single channels for clarity. l–l″, PI Ms neurons do not co-express Ilp2, used as a marker of insulin-producing neurons. m–m″, PI Ms neurons do not co-express Dh44-Gal4, used as a marker of Diuretic Hormone 44-producing neurons. n–n″, PI Ms neurons do not co-express Mip-Gal4, used as a marker of Myoinhibiting peptide precursor-producing neurons. o–o″, Co-expression between Ms and Mip-Gal4 in three out of the five HCG Ms-expressing neurons. Phalloidin was used to label gut muscles (in blue). p–p″, A subset of PI Ms neurons co-express Taotie-Gal4; other Taotie-Gal4-positive PI neurons are Ms-negative. In the HCG, Taotie-Gal4 expression is only apparent inconsistently in one Ms neuron (data not shown). q–q″, PI Ms neurons do not co-express Dsk-Gal4, used as a marker of Drosulfakinin-producing neurons. Scale bars: b, d′, f–h and k–k′ = 50μm; i–j″, l–o″ and q–q″ = 25 μm, b (inset), c, d, p–p″ = 20 μm and k (inset) = 10 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions.
Extended Data Fig. 4 Ms neuron regulation of crop enlargement.
a–a′, Validation of MsΔ mutant using anti-Ms staining shown in green; PI is highlighted by dashed lines. a, Lack of Ms staining in the PI of Ms mutants (MsΔ/Df(3R)Exel6199). Ms staining is apparent in the PI of Df(3R)Exel6199 (a′) and MsΔ (a″) heterozygous control flies. b, Quantifications of crop area in ad libitum-fed flies upon Ms-Gal4-driven TrpA1 expression (4 h at the permissive temperature), showing these have significantly larger crops relative to UAS and Gal4 controls. c, Quantifications of crop area in starved–refed flies upon Ms-Gal4-driven Kir2.1 expression (temporally confined with tub-Gal80TS), showing that these have significantly smaller crops relative to UAS and Gal4 controls. d–e″, Effect of neuronal activation and Ms downregulation on Ms levels in PI neurons. Thermogenic activation of Ms neurons in ad libitum fed flies depletes Ms peptide (in red) from Ms neuron cell bodies in the PI (d) compared to UAS (d′) and Gal4 (d″) controls. Adult-specific Ms downregulation in Ms neurons of starved-refed flies results in reduced Ms staining (red) in PI neurons (e), compared to UAS (e′) and Gal4 (e″) controls. f–i, Effect of Ms loss-of-function and adult-specific Ms neuron inactivation on crop expansion and shape, upon starvation-refeeding in mated females. f, Quantifications of crop area reveal that Ms neuron inactivation results in smaller crops relative to Ms mutant or w1118, UAS and Gal4 controls. g, Representative crop images of genoytpes quantified in f. h, Quantifications of crop roundness reveal that crops are less round upon Ms neuron inactivation or in Ms mutant compared to w1118, UAS and Gal4 controls. i, PCA of landmark position variation along the crop outline, showing that crop shapes are distinct between Ms mutant (red), Ms neuron inactivation (yellow) and w1118 (grey), being more similar between Ms mutant and w1118, as highlighted by partial overlap of their 95% confidence ellipses. Wireframe deformation grids are shown to illustrate the mininum and maximum shape deviations as compared to the mean shape along each PC axis. j, k, Effect of Ms neuron activation on crop expansion in Ms mutant background, upon starvation-refeeding in mated females. j, Quantifications of crop area show that activation of Ms neurons by Ms-Gal4-driven TrpA1 expression results in larger crops relative to activation of Ms neurons by MsTGEM-driven TrpA1 expression in an heteroallelic mutant background, as well as relative to Ms mutant or UAS and Gal4 controls. k, Representative crop images of flies with the genoytpes quantified in j. l, m, Effect of Ms and Taotie neuron activation on crop enlargement, upon starvation in mated females. l, Quantification of crop area shows that activation of either Ms neurons or Taotie neurons results in larger crops compared to respective Gal4 controls and UAS control, even in the absence of food. m, Representative crop images of genotypes quantified in l. Scale bars: a–a′ = 10 μm, d–e″ = 25 μm, g, k, m = 500 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 5 Expression of Ms receptors and their regulation of crop enlargement.
a, FB1.1-derived EGFP reveals MsR1 expression in the crop muscles and nervous system, including nerves innervating the crop, hindgut and rectal ampulla. In this and subsequent panels, muscles are labelled with phalloidin (in blue). b–b″, Co-expression of MsR1 mRNA stained with single-molecule RNA fluorescence in situ hybridization (b, b′, in red) and FB1.1-derived EGFP driven by MsR1TGEM-Gal4 (b, b″, in green) is observed in crop muscles. Muscle nuclei are shown in blue with DAPI; single channels are shown for clarity. c, Detail of the HCG and corpora cardiaca (CC); the latter is extensively innervated by MsR1-expressing neuronal projections. d, FB1.1-derived EGFP reveals MsR1 expression in neurons innervating the female reproductive system, but not in its muscles. e, FB1.1-derived EGFP reveals MsR1 expression in heart-innervating neurons, but not in heart muscles. f, Higher magnification image of the central brain; nuclear GFP reveals broad MsR1 expression in neurons including the PI Ms neurons shown with Ms staining (in red). g, A subset of 2–3 MsR1-positive neurons in the HCG co-express Ms. h, Nuclear GFP driven from MsR1TGEM-Gal4 reveals co-expression of MsR1 and Akh (in red) in CC cells. i, Single-molecule fluorescence in situ hybridization of MsR1 and MsR2 mRNAs in crop muscles; MsR1 (in green) is more readily detected than MsR2 (in red). Muscle cell nuclei are shown in blue by DAPI staining. The MsR1 expression described in a–h is consistent with transcriptomics data92,93. i′ and i″ show single MsR1 or MsR2 channels for clarity. j–j′', Validation of adult-specific MsR1 knockdown in visceral muscles (vmTS > MsR1 KD). Panels show high-magnification images of crop muscles. MsR1 mRNA expression is visualized by single-molecule RNA fluorescence in situ hybridization (in green) in Gal4 (j′) and UAS (j″) controls, but it is reduced or absent when MsR1 is downregulated from crop muscles (vm-Gal4TS > MsR1 KD) (j). k, Quantifications of crop area in starved–refed flies upon downregulation of MsR1 in visceral muscles, showing that crop size is significantly reduced upon MsR1 downregulation compared to UAS and Gal4 controls. l, A similar reduction in crop area is also quantified upon MsR1 downregulation specifically in crop muscles using a different driver line (MsR1crop > MsR1RNAi). MsR1crop-Gal4 is MsR1-Gal4, nsyb-Gal80, in which MsR1-Gal4 neuronal expression is prevented using the pan-neuronal nsyb-Gal80 driver, rendering it a crop muscle-specific driver. m–o″, Effect of crop-muscle-specific downregulation of MsR1 on crop size. m, Quantifications of crop area in starved–refed mated females show that crop-specific downregulation of MsR1 (MsR1crop > MsR1RNAi) results in reduced crop areas compared to Gal4 and UAS controls, similar to Ms neuron inactivation (Ms > Kir2.1). n–o″, Representative crop phenotypes of flies with the genotypes quantified in m. p, Quantifications of crop area upon visceral muscle-specific MsR1 and MsR2 downregulation, showing that MsR1 downregulation, but not MsR2 downregulation, results in reduced crop sizes, as compared to UAS and Gal4 respective controls. q, Quantifications of crop area in starved–refed mated females show that heteroallelic MsR1TGEM/DfAprt-32 mutants have reduced crop areas relative to w1118 or heterozygous controls. r, Representative crop images from genotypes quantified in q. s, Validation of MsR1 mutation and MsR1 fluorescence in situ hybridization signal specificity. MsR1 mRNA (green) is absent from the crop muscle cells of MsR1TGEM mutants, and apparent in w1118 control flies. Scale bars: b–b″, f–j″ and s = 10 μm; a, c–e = 50 μm; r = 500 μm; n–o″ = 1 mm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 6 Post-mating modulation of Ms neurons.
a, b, Analysis of Ms neuron crop terminals in virgin and mated females. Neither the number of axonal branches (a) nor their diameter (b) is significantly different between virgin and mated females. c, Quantifications of Ms staining levels in the cell bodies of PI neurons of wild-type, ad libitum-fed males, virgin females and mated females. Mated females have less Ms peptide than virgin females or males; virgin females have less peptide than males. d–h, Comparison of Ms peptide levels in the cell bodies of PI neurons in fed versus starved virgin and mated females. Representative images of Ms staining in the cell bodies of the PI neurons of fed virgin females (d), starved virgin females (e), fed mated females (f) and starved mated females (g). h, Quantification of Ms staining in the cell bodies of PI neurons shows that Ms levels are reduced in mated females compared to virgins, irrespective of fed or starved status. i, RT-qPCR expression data for Ms transcript levels in the brain of ad libitum-fed, control males (grey column), virgin females (pink column) and mated females (red column). No significant differences are apparent between groups. j–l, CaLexA-based assessment of mating-triggered changes in PI Ms neuronal activity, achieved by adult- and Ms-confined CaLexA expression (MsTS > CaLexA). Representative images of ad libitum-fed, wild-type virgin (j, j′), and mated females (k, k′) are shown. Ms neurons are labelled with anti-Ms antibody (in red) and CaLexA channel is shown as a single channel (in green) for clarity. l, Quantification of CaLexA-derived GFP-positive cells in PI Ms neurons of virgin (pink box) and mated (red box) females shows that fewer cells are CaLexA-positive in virgin compared to mated females; each data point corresponds to a different brain. m, n, Quantification of baseline GCaMP fluorescence (corrected for background) (m) and amplitude of GCaMP fluorescence oscillations (n) in the cell bodies of PI Ms neurons of virgin females (pink box) or mated females (red box). Each data point corresponds to an individual cell measurement. Higher GCaMP signal and reduced oscillation amplitude are detected in mated females. o, Crop area quantifications in wild-type, ad libitum-fed males, virgin females and mated females. The crop of mated females is bigger than that of virgin females or males. p, q, Effects of sex and mating status on Ms signalling contribution to crop size. p, Quantification of crop area upon adult-specific downregulation of MsR1 in visceral muscles shows that it is significantly reduced in mated females but not in males or virgin females, as compared to respective controls. q, Representative crop images of genotypes quantified in m. Scale bars: d-g, j-k′ = 20 μm; q = 500 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 7 Ecdysone modulation of Ms neurons and crop size.
a–a′, Expression of EcR in PI Ms neurons. Ms (antibody labelled in green) is co-expressed with EcR (labelled in red with an antibody that recognizes all EcR isoforms) (a). EcR staining is shown as a single channel below for clarity (in red) (a′). b–d, Ecdysone effect on Ms levels in PI neurons. Representative images show comparable Ms levels upon expression of EcRDN in virgin females (b) relative to UAS (b′) and Gal4 (b″) controls. Fluorescence signals are pseudo-coloured; high to low intensity is displayed as warm (yellow) to cold (blue) colours. c, Quantification of Ms staining intensities in PI neurons of virgin females upon expression of EcRDN shows comparable levels to UAS and Gal4 controls. d, Quantification of Ms staining intensities in PI neurons of mated females upon expression of EcRDN shows increased Ms levels relative to UAS and Gal4 controls. e, Quantification of crop area in starved-refed mated females reveals smaller crops upon adult- and Ms neuron-specific EcR downregulation compared to UAS and Gal4 controls. f–j, Classification of crop size upon expression of EcRDN (f, g), EcR-BI downregulation (h, i) or EcR (all isoforms) downregulation (j) in starved–refed female flies. The distribution of crop sizes does not significantly differ from that of UAS and Gal4 controls in virgin females (f, h, j). In mated females, the distribution shifts towards smaller crop sizes, relative to UAS and Gal4 controls (g, i). Ranked data are displayed as percentages. Scale bars, 20 μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 8 Bursicon modulation of Ms neurons.
a, Co-expression of Burs (a′, in red), Pros (a″, in white) and GFP driven by Tkg-Gal4 (a‴, in green) in midgut enteroendocrine cells of mated females. b, Quantifications of Pros-positive midgut cells show increased enteroendocrine cell number in mated females relative to virgins. Flies were starved for 22 h to increase Burs staining in the enteroendocrine cell bodies37. Single channels for each marker are shown for clarity. c, Quantifications of enteroendocrine cells of mated females labelled by Tkg-Gal4-driven EGFP and Burs staining (such as that shown in a). More Tkg-Gal4-positive than Burs-positive enteroendocrine cells are apparent. The majority of Burs-positive enteroendocrine cells are Tkg-Gal4-positive. d, e, Co-expression of rkTGEM (driving FB1.1, in green) and Ms peptide (in red) is shown in brain and VNC neurons (d), and in the HCG ganglion (e). f–f′, Co-expression of rkTGEM (driving FB1.1-derived EGFP, in green) and Ms peptide (in red) is apparent in brain PI neurons. f′, Ms staining is shown as a single channel for clarity. g–g′, Co-expression of Ms-Gal4 (driving FB1.1-derived EGFP, in green) and rk mRNA (stained with fluorescence in situ hybridization, in red) is apparent in brain PI neurons. g′, rk fluorescence in situ hybridization signal is shown as a single channel for clarity. h, Co-expression of rkTGEM (driving FB1.1-derived EGFP, in green), Ms peptide (in white) and EcR (in red) is apparent in brain PI neurons. i, Co-expression of Taotie-Gal4 (driving FB1.1-derived EGFP, in green) and EcR (in red) is apparent in brain PI neurons. Nuclei are stained with DAPI (in blue). j–j′, Co-expression of Taotie-Gal4 (driving FB1.1-derived EGFP, in green) and rk mRNA (stained with fluorescence in situ hybridization, in red) is apparent in brain PI neurons. Nuclei are stained with DAPI (in white). j′, rk mRNA fluorescence in situ hybridization signal is shown as a single channel for clarity. k–m, rk regulation of Ms levels in PI neurons. Representative images show similar Ms staining signal upon adult-specific rk downregulation in virgin females (k) relative to UAS (k′) and Gal4 (k″) controls. Fluorescence signals are pseudo-coloured; high to low intensity is displayed as warm (yellow) to cold (blue) colours. l, Quantification of Ms staining intensities in PI neurons of virgin females upon adult-specific rk downregulation showed comparable levels to UAS and Gal4 controls. m, Quantification of Ms staining intensities in PI neurons of mated females upon adult-specific rk downregulation showed increased Ms levels relative to UAS and Gal4 controls. n, Quantification of the amplitude of GCaMP oscillations in PI neurons of mated females shows that downregulation of EcR and rk in Ms neurons significantly increases the amplitude of calcium signal. o, Quantification of GCaMP baseline fluorescence levels in PI neurons of mated females reveals that downregulation of EcR in Ms neurons significantly reduces GCaMP levels, whereas downregulation of rk increases GCaMP levels, both relative to expression of EGFP. Hence, calcium oscillations become virgin-like upon both EcR or rk downregulation, whereas their effects on overall calcium fluorescence are different. Scale bars = 20 μm apart from a–a″, d, e = 50μm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 9 Post-mating modulation of crop enlargement by Burs and ecdysone.
a–a′, Classification of crop size upon rk downregulation in Ms neurons of starved–refed female flies. The distribution of crop sizes does not significantly differ from that of UAS and Gal4 controls in virgin females (a). In mated females, the distribution shifts towards smaller crop sizes, relative to UAS and Gal4 controls (a′). Ranked data are displayed as percentages. b–e, Effect of Burs downregulation from enteroendocrine cells on crop enlargement in virgin (b, d) and mated (c, e) females. Representative crop images of ad libitum-fed flies virgin females show that crop size is not visibly changed upon downregulation of Burs in Pros-expressing enteroendocrine cells (b) relative to UAS (b′) and Gal4 (b″) controls. By contrast, in mated females, crops are less expanded (c), relative to UAS (c′) and Gal4 (c″) controls. Quantifications of crop area of genotypes shown in b–b″ and c–c″ are shown in d and e respectively. f–h, Thermogenic activation of Tkg-Gal4-positive cells (which include Burs-positive enteroendocrine cells but also a very small subset of neurons outside the PI, not shown) results in significantly reduced Ms levels in the cell bodies on PI neurons of virgin females, relative to UAS and Gal4 virgin controls. f–g″, Representative images of Ms staining in PI neurons of the genotypes quantified in h. Reduced Ms staining is apparent in PI neurons of virgin females upon activation of Tkg-Gal4-positive cells (f) relative to UAS (f′) and Gal4 (f″) virgin controls. The difference between activated (g) versus control (g′, g″) flies is not apparent when female flies are mated (presumably because more Ms peptide has been released in controls). Fluorescence signals are pseudo-coloured; high to low intensity is displayed as warm (yellow) to cold (blue) colours. i, j, Effect of gut hormone release from enteroendocrine cells on crop enlargement. Representative crop images of ad libitum-fed female flies show that crop size is increased upon thermogenic activation of Tkg-Gal4-positive cells (i) relative to UAS (i′) and Gal4 (i″) controls, quantified in j. We note that the Tkg-Gal4-positive cells include most Burs-positive enteroendocrine cells as well as a very small subset of central neurons outside the PI (not shown). k, l, Effect of ecdysone and Burs signalling in Taotie neurons on crop enlargement after mating. Representative crop images of starved–refed mated females show that, relative to the UAS control (k), downregulation of EcR (k′) or rk (k″) results in visibly smaller crops. Quantifications of crop area of genotypes shown in k–k″ are shown in l. m, Schematic summary of key findings. Circulating levels of Bursicon and Ecdysone increase after mating. Ecdysone and Burs signal via their receptors to Ms neurons, change their neural activity, leading to crop enlargement. Scale bars: f–g″ = 20 μm; b–c″, i–i″ = 500 μm; k–k″ = 1 mm. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 10 Regulation of food intake, fecundity and fertility by Ms neurons.
a, b, Mated females increase their food intake. Both the amount of ingested dye-laced food (a) and the number of sips per fly (b) are increased in wild-type mated females relative to virgins. c–e, Regulation of food intake by MsR1 downregulation in crop muscles. Quantifications of ingested dye show that downregulation of MsR1 in the visceral muscles of starved-refed virgin females results in similar food intake relative to UAS and Gal4 controls (c), whereas downregulation of MsR1, but not MsR2, in mated females, results in reduced food intake relative to UAS and Gal4 controls (d). e, Quantification of the number of sips per fly shows that downregulation of MsR1 specifically in crop muscles using an independent driver line also reduces food intake relative to Gal4 and UAS controls in starved–refed mated females. f, Quantifications of ingested dye-laced food show that downregulation of EcR in Ms neurons of starved–refed virgin females does not significantly affect food intake when compared to Gal4 and UAS controls. g, Similarly, quantifications of ingested dye-laced food show that downregulation of Burs in Pros-expressing enteroendocrine cells of starved–refed virgin females does not significantly affect food intake when compared to Gal4 and UAS controls (g). h, In the model, food ingression from the oesophagus is driven by crop enlargement, which is assumed to be linear during sips and constant in between sips. The observed increase in food intake in mated females compared to virgins can be explained by a decrease in negative pressure from −0.8 kPa to −1.3 kPa (increased suction), leading to an increased intake during sips. See Source Data for crop morphometry and FlyPad quantifications used for this crop fluid dynamics model. i, j, Thermogenic activation of Ms neurons (Ms > TrpA1) for 4 h before the transfer of flies from undyed to dye-laced food reduces the mean amount of ingested dye during the course of 1 h (i), and reduces the mean number of sips per fly over 1 h of feeding (j) relative to Gal4 and UAS controls. k, l, Concurrent thermogenic activation of Ms neurons during feeding of dye-laced food increases the mean amount of ingested dye during the course of 1 h (k), but has no effect on the mean number of sips per fly over 1 h of feeding (l′) relative to Gal4 and UAS controls. m, n, Effect of neuronal activation on the regulation of food intake by Taotie-Gal4-positive neurons. Quantification of ingested dye-laced food shows that thermogenic activation of Taotie neurons for 4 h before the switch from undyed to dye-laced food reduces the amount of ingested dye relative to Gal4 and UAS controls over the course of 1 h (m). By contrast, concurrent activation during feeding of such food increases the amount of ingested dye relative to Gal4 and UAS controls over the course of 1 h (n). o, p, Effect of Ms signalling to crop muscles on fecundity and fertility. o, Quantification of eggs laid in 24 h by mated females shows that MsR1 downregulation specifically in crop muscles results in significantly fewer eggs laid after 4 days relative to UAS and Gal4 controls. p, Quantification of adult progeny produced from a 24-h period of egg laying by mated females, shows that MsR1 downregulation in visceral muscles results in significantly fewer progeny relative to UAS and Gal4 controls. Sip number measurements were taken over 1 h of feeding. See Supplementary Information for a list of full genotypes, sample sizes and conditions. In all box plots, line: median; box: 75th–25th percentiles; whiskers: minimum and maximum. All data points are shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Supplementary information
Supplementary Information
This file contains a list of full genotypes and sample sizes (numbers of animals/repeats per experiment) and experimental conditions.
Video 1
In vivo dynamics of crop and midgut enlargement. Dynamics of crop and midgut enlargement in a virgin female fly fed dye-laced liquid yeast/sucrose food (concentrations as per our standard food) through a fine-tipped capillary. Labelled pauses indicate when food first enters the crop and the midgut. Time is 4x faster.
Video 2
In vivo imaging of post-mating changes in calcium dynamics in Ms neurons. Mating increases GCaMP6 fluorescence levels and reduces the amplitude of GCaMP6 oscillations in the PI Ms neurons of adult female flies in vivo. Two representative videos are shown (see Fig. 2c,d for snapshots and Extended Data Fig. 6m,n for quantifications of GCaMP6 oscillation amplitude and fluorescence levels). Time is 4x faster.
Video 3
In vivo imaging of calcium dynamics in Ms neurons following EcR or rk downregulations. The amplitude of GCaMP6 oscillations in the PI Ms neurons is increased (i.e. more virgin-like) in the PI Ms neurons of adult, mated female flies in vivo when compared to that of control adult, mated female flies. Three representative videos are shown (see Extended Data Fig. 8n,o for quantifications of GCaMP6 oscillation amplitude and fluorescence levels). Time is 4x faster.
Source data
Rights and permissions
About this article
Cite this article
Hadjieconomou, D., King, G., Gaspar, P. et al. Enteric neurons increase maternal food intake during reproduction. Nature 587, 455–459 (2020). https://doi.org/10.1038/s41586-020-2866-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-020-2866-8
This article is cited by
-
Male manipulation impinges on social-dependent tumor suppression in Drosophila melanogaster females
Scientific Reports (2024)
-
Drosophila activins adapt gut size to food intake and promote regenerative growth
Nature Communications (2024)
-
Metabolic and feeding adjustments during pregnancy
Nature Reviews Endocrinology (2023)
-
Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson’s disease in Drosophila
Translational Neurodegeneration (2022)
-
Food craving-like episodes during pregnancy are mediated by accumbal dopaminergic circuits
Nature Metabolism (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
