Malpighian tubule development in the red flour beetle (Tribolium castaneum) Benedict King 1 , Barry Denholm * Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK article info Article history: Received 17 June 2014 Accepted 24 August 2014 Available online xxx Keywords: Malpighian tubule Organogenesis Insect excretory system Tribolium Beetle Drosophila Cryptonephridial system abstract Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent- extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types e principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryp- tonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty e the cryptonephridial system e arose during organ evolution. Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved. 1. Introduction For most insects the Malpighian tubule (MpT) is the principal organ of excretion. It controls water, ion and acid/base balance, and removes toxins and metabolic wastes. MpTs are simple, single cell- layered epithelial tubes equipped for the transport of water, ions and toxins, most usually from the haemolymph into the tubule lumen. The contents of the lumen are modified before being expelled into the hindgut, where further modification can also take place (Beyenbach et al., 2010; Denholm, 2013). Although their general form is similar across the insects, MpTs can vary substantially in size and number, ranging for example from the two long pairs of tubules found in Drosophila and in the blood-sucking bug Rhodnius, to the few hundred shorter tubules found in locusts and crickets (Skaer, 1992; Skaer et al., 1990; Hazelton et al., 1988; Wessing and Eichelberg, 1978; Snodgrass, 1935; Wigglesworth, 1939, 1931). In some species the MpTs have been modified for specialised functions. The rectal complex or cryptonephridial system, which is particularly prevalent in Co- leopterans and in the larvae of Lepidopterans, is one example (Fig. 1A) (Ramsay, 1964; Grimstone et al., 1968; Ramsay, 1976; Saini, 1964). Here, the distal portions of the tubules form an intimate association with the rectum in a system that allows water to be drawn osmotically from the contents of the rectum back into the tubule, and from there recycled back to the haemolymph from the proximal tubule. The cryptonephridial system is considered an adaptation to minimize water loss. Drosophila melanogaster stands alone as the one insect in which genetic and molecular development is known in detail. Over the last few decades we have learned how this organ is specified, how it grows and is shaped, and is positioned within the body of the an- imal (Skaer, 1989; Ainsworth et al., 2000; Kerber et al., 1998; Sudarsan et al., 2002; Denholm et al., 2003; Bunt et al., 2010; Abbreviations: BMP, bone morphogenetic protein; CE, convergent-extension; DAPI, 4 0 ,6-diamidino-2-phenylindole; D-P, disto-proximal; dpERK, di-phosphory- lated ERK; Dpp, decapentaplegic; EGF, epidermal growth factor; GFP, green fluo- rescent protein; MpT, Malpighian tubule; PBS, phosphate buffered saline; PC, principal cell; SC, stellate cell; SD, standard deviation; Spi, Spitz; Tc, Tribolium castaneum; TC, tip cell; Tio, Tiptop; Tsh, Teashirt. * Corresponding author. Present address: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, Edinburgh, Scotland EH8 9XD, UK. Tel.: þ44 (0)131 6509880. E-mail addresses: king0393@uni.flinders.edu.au (B. King), Barry.Denholm@ed. ac.uk (B. Denholm). 1 Present address: School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia 5001, Australia. Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd http://dx.doi.org/10.1016/j.asd.2014.08.002 1467-8039/Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved. Arthropod Structure & Development xxx (2014) 1e9 Please cite this article in press as: King, B., Denholm, B., Malpighian tubule development in the red flour beetle (Tribolium castaneum), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.08.002