NHA1 is a cation/proton antiporter essential for the water-conserving functions of the rectal complex in Tribolium castaneum

Abstract

AbstractMore than half of all extant metazoan species on earth are insects. The evolutionary success of insects is intrinsically linked with their ability to osmoregulate, suggesting that they have evolved unique physiological mechanisms to maintain water balance. In beetles (Coleoptera)—the largest group of insects—a specialized rectal (‘cryptonephridial’) complex has evolved that recovers water from the rectum destined for excretion and recycles it back to the body. However, the molecular mechanisms underpinning the remarkable waterconserving functions of this system are unknown. Here, we introduce a transcriptomic resource, BeetleAtlas.org, for red flour beetle Tribolium castaneum, and demonstrate its utility by identifying a cation/H+ antiporter (NHA1) that is enriched and functionally significant in the Tribolium rectal complex. NHA1 localizes exclusively to a specialized cell type, the leptophragmata, in the distal region of the Malpighian tubules associated with the rectal complex. Computational modelling and electrophysiological characterization in Xenopus oocytes show that NHA1 acts as an electroneutral K+/H+ antiporter. Furthermore, genetic silencing of Nha1 dramatically increases excretory water loss and reduces organismal survival during desiccation stress, implying that NHA1 activity is essential for maintaining systemic water balance. Finally, we show that Tiptop, a conserved transcription factor, regulates NHA1 expression in leptophragmata and controls leptophragmata maturation, illuminating the developmental mechanism that establishes the novel functions of this cell. Together, our work provides the first insights into the molecular architecture underpinning the function of one most powerful water-conserving mechanisms in nature, the beetle rectal complex.Significance StatementBeetles are the largest group of insects, inhabiting a wide range of habitats on earth. Unique adaptations in overcoming water stress is critical to their success, yet the mechanisms underpinning this ability are unknown. Using genetics, electrophysiology, imaging and behavioral studies we show that a cation/H+ (NHA1) transporter is exclusively localized to specialized cell type, the leptophragmata, in the Malpighian tubules associated with the rectal complex. Ion transport functions of NHA1 in leptophragmata underpin the movement of water from the rectum, from where it would be destined for excretion, to the Malpighian tubule and then recycled back to the body. This water recovery capability of rectal complex is essential for maintaining systemic water balance in beetles. This work provides the first insight into to the molecular architecture of one of most powerful water-conservation mechanisms in biology, and provides an important clue to the ecological and evolutionary success of the beetles.