The cryptonephridial/rectal complex: an evolutionary adaptation for water and ion conservation

Abstract

Arthropods have integrated digestive and renal systems, which function to acquire and maintain homeostatically the substances they require for survival. The cryptonephridial complex (CNC) is an evolutionary novelty in which the renal organs and gut have been dramatically reorganised. Parts of the renal or Malpighian tubules (MpTs) form a close association with the surface of the rectum, and are surrounded by a novel tissue, the perinephric membrane, which acts to insulate the system from the haemolymph and thus allows tight regulation of ions and water into and out of the CNC. The CNC can reclaim water and solutes from the rectal contents and recycle these back into the haemolymph. Fluid flow in the MpTs runs counter to flow within the rectum. It is this countercurrent arrangement that underpins its powerful recycling capabilities, and represents one of the most efficient water conservation mechanisms in nature. CNCs appear to have evolved multiple times, and are present in some of the largest and most evolutionarily successful insect groups including the larvae of most Lepidoptera and in a major beetle lineage (Cucujiformia + Bostrichoidea), suggesting that the CNC is an important adaptation. Here we review the knowledge of this remarkable organ system gained over the past 200 years. We first focus on the CNCs of tenebrionid beetles, for which we have an in-depth understanding from physiological, structural and ultrastructural studies (primarily in Tenebrio molitor), which are now being extended by studies in Tribolium castaneum enabled by advances in molecular and microscopy approaches established for this species. These recent studies are beginning to illuminate CNC development, physiology and endocrine control. We then take a broader view of arthropod CNCs, phylogenetically mapping their reported occurrence to assess their distribution and likely evolutionary origins. We explore CNCs from an ecological viewpoint, put forward evidence that CNCs may primarily be adaptations for facing the challenges of larval life, and argue that their loss in many aquatic species could point to a primary function in conserving water in terrestrial species. Finally, by considering the functions of renal and digestive epithelia in insects lacking CNCs, as well as the typical architecture of these organs in relation to one another, we propose that ancestral features of these organs predispose them for the evolution of CNCs.

Keywords: Coleoptera; Tenebrionidae; Tribolium castaneum; arid environment; arthropod; beetle; countercurrent; cryptonephridium; insect; renal system.

Fig. 1

Cryptonephridial complex (CNC) organisation and function. (A) Schematic of gut and Malpighian tubule (MpT) organisation of Tribolium castaneum adult (a similar organisation is seen in the larval stage). The proximal, open ends of the MpTs insert into the boundary of the midgut and hindgut. As for many species of Coleoptera, Tribolium has a CNC, in which the distal segments of the MpTs lie on the external surface of the rectum, and are ensheathed with a perinephric membrane (PNM) tissue, a cross section of which is shown. All MpTs also have a region which lies freely in the haemolymph (part of one is shown for simplicity, compare with Fig. 4D). (B) In adult Drosophila melanogaster, MpTs are free within the haemolymph, and are not bound to the rectum (this is also the case in the larval stage). Similar organisations are found in diverse insects including most Diptera, and this is considered the ancestral state. (C) Larval Trichoplusia ni has a CNC organisation typical of most larval Lepidoptera, which has evolved independently from the CNCs of Coleoptera, but shows some remarkable similarities in organisation. Modified from O'Donnell & Ruiz‐Sanchez (2015). Generally in Lepidoptera species, the MpTs fold back on themselves within the CNC to form an inner and outer layer (Henson, ; Ishimori, 1924), not shown for simplicity. (D) A countercurrent organisation underpins the function of the CNC in beetles such as Tribolium. The system is insulated from water and ion exchange with the haemolymph by the PNM, with only the specialised leptophragma (lp) cells having access to the haemolymph. Potassium chloride is transported into the MpT lumen via the lp, driving a flux of water from the rectal contents into the MpTs. This fluid flows into the free portion of the MpTs where reabsorption of ions into the haemolymph can drive the return of water into the animal. The fluid flow in the rectum and MpTs are in opposite directions, meaning the osmolarity is highest at the posterior of the system (right), which maximises its ability to extract water, and minimises water loss from the animal. The system also enables the gain of water from humid air entering via the anus. (E) Schematic cross section through the CNC, as seen in Tenebrio and Tribolium. O‐ and I‐ PNM are the outer and inner layers, respectively, of the PNM. The lp of the MpTs lie beneath thin blisters (bl). Longitudinal and circular muscles (LM and CM) surround the rectal epithelium (RE). (F) A cross section of part of the CNC, corresponding to the boxed area in E. Vacuolar ATPase (V‐ATPase) pumps protons (H⁺) into the MpT lumen. Na⁺, K⁺/H⁺ antiporter 1 (NHA1) appears to act as a K⁺/H⁺ antiporter, using these protons to drive the transport of K⁺ into the MpT lumen via the lp. Along with Cl⁻, thought to flow along the electrochemical gradient established by K⁺ transport, this generates high osmolarity surrounding the rectum, drawing water from the rectal contents. The transcription factor, Dachshund (Dac), represses urinate receptor (Urn8R) expression, perhaps by binding and repressing another transcription factor, Tiptop (Tio), in the nucleus (nu). This may prevent the lp from increasing secretion in response to diuretic hormone 37 (DH₃₇) and 47 (DH₄₇) signalling. (G) Cross section through the free region of a MpT showing a secondary cell (SC) and principal cell (PC). SCs in this region express a hormone receptor (Urn8R), likely under control of the transcription factor Tio. In response to low osmolarity, the brain releases DH₃₇ and DH₄₇. These bind to the Urn8R receptor, upregulating K⁺ transport, and driving fluid secretion into the MpT lumen.

Fig. 2

Embryonic development of the cryptonephridial complex (CNC) in Tribolium. (A) Six Malpighian tubules (MpTs) bud out from the end of the developing hindgut/rectum. The hindgut/rectum expresses the fibroblast growth factor 8 (FGF8) ligand. In Tribolium, a single FGF receptor (FGFR) gene is expressed as distinct isoforms, which receive the different FGF ligands. The FGFR isoform for FGF8 is expressed in a posterior population of mesoderm. FGF8 signalling recruits the posterior mesoderm onto the hindgut/rectum. (B) A second FGF ligand, Branchless (Bnl) becomes expressed in the region of posterior mesoderm now surrounding the posterior end of the developing rectum. Tip cells, which guide the growing MpTs, express the FGFR isoform for Bnl. This pathway guides the MpTs to grow into the posterior mesoderm, closely surrounding the hindgut/rectum. The epidermal growth factor (EGF) ligand spitz signals from the distal end of the MpT, and likely drives and orients MpT elongation. (C) FGF8 continues to be expressed in the hindgut/rectum, but also switches on in the MpTs, and is likely responsible for some of the posterior mesoderm migrating over the MpT surface. (D) The posterior mesoderm coats the MpTs and hindgut, and will differentiate into the perinephric membrane, and muscle cells, for example those that form a mesh around the free MpTs. The distal ends of the MpTs have established their countercurrent organisation with the rectum, and become convoluted, so entirely surrounding the rectum.

Fig. 3

Evolutionary occurrence of the cryptonephridial complex (CNC)/rectal complex in Coleoptera and Lepidoptera. (A) Phylogenetic tree of Coleoptera, showing reports of CNC or rectal complex presence (green circles) or absence (magenta circles). The earliest likely origin of the CNC/rectal complex is indicated by the green arrow. The tree is drawn according to McKenna et al. (2019) and represents a ~300‐million‐year evolutionary period. Its presence in the indicated groups means that a CNC/rectal complex is likely to occur in ~190,000 known beetle species, not counting Scarabaeoidea (based on species numbers in Hunt et al., 2007). A rectal complex has likely evolved independently in Scarabaeoidea (light orange) (Beaven et al., 2024a ). The suborder Polyphaga and the clade Phytophaga are indicated. (B) Phylogenetic tree of Lepidoptera, annotated as in A. The tree is drawn according to Kawahara et al. (2019) and represents a ~300‐million‐year evolutionary period. CNC/rectal complex presence in the indicated groups means that it is likely to occur in ~160,000 known species of Lepidoptera (based on species numbers in Regier et al., 2009). The clade Ditrysia is indicated. Further details of species and variations in the observed CNC/rectal complex structures are provided in Table S1.

Fig. 4

Architectures of insect digestive/renal organs. (A) Organisation seen in larval Keroplatus testaceus (modified from Stammer, 1932), a dipteran with a rectal complex. (B) Organisation seen in adults of Anthrenus species (Saini, ; R. Beaven & B. Denholm, unpublished observations), with a laterally displaced cryptonephridial complex (CNC). Note that the cryptonephridial Malpighian tubules (MpTs) are obscured by the perinephric membrane (PNM). A similar organisation exists in larvae (Mobüsz, 1897). lp, leptophragma. (C) Organisation seen in Drosophila adults, a species without a rectal complex. Calcium transport from the midgut contents to the distal regions of the anterior MpTs is indicated. Approximately equivalent positions are also seen in larvae. (D) Organisation of adult Tribolium, a species with a CNC, showing the full extent of the free MpT region for one tubule. Note that the perirectal MpTs are obscured by the PNM, but are illustrated in Fig. 1A. A very similar organisation is seen in larvae. (E) Organisation seen in Tomaspis (Cercopidae, Hemiptera) (modified from Wigglesworth, 1974b ), a species with a filter chamber. Arrows show the direction of flow of gut contents. All images are coloured according to labels in D and E. Note that in A–D the most anterior gut structures are omitted. See Fig. 1C for a comparison with lepidopteran larva.