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

Abstract

More 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 water-conserving 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.

DrosophilaFog/Cta and T48 pathways have overlapping and distinct contributions to mesoderm invagination

“…Although the involvement of T48 pathway before mesoderm invagination is not known in other insects, Fog/Cta pathway is required for the blastoderm formation in multiple insect species including Tribolium (Benton et al ., 2019). Publicly available Tribolium transcriptome data shows the enrichment of t48 in female gonad albeit in less degree compared to fog (Naseem et al ., 2023). Given that T48 facilitates Fog/Cta pathway during mesoderm invagination in Tribolium (Benton et al ., 2019), it is conceivable that T48 pathway also plays a role in blastoderm formation and/or oogenesis in other basal insects.…”

Something old, something new: the origins of an unusual renal cell underpinning a beetle water-conserving mechanism

“…To test this, we dissected adult CNCs and used an antibody to detect Tiptop protein, in combination with counter-staining for F-actin, allowing us to identify the leptophragmata by position and cellular morphology. In Tribolium and the related Tenebrionid beetle, Tenebrio molitor, the perinephric membrane ensheaths the CNC but is punctuated with small windows under which the leptophragmata sit (5,6,14,16).The outer perinephric membrane, which stains strongly for F-actin, and the windows within it, can readily be identified. The leptophragmata are also apparent, both by their position beneath the window and by the distinctive bar-shaped F-actin structure contained within them.…”

“…Using the model insect species the red flour beetle ( Tribolium castaneum ) we have shed light on the molecular mechanisms underpinning leptophragmata function. We identified a cation/proton antiporter, NHA1, in the leptophragmata, which likely uses a proton gradient to transport potassium into the CNC (16). In turn, chloride is considered to follow along the electrochemical gradient established by potassium transport (7, 8).…”

“…We previously demonstrated that Tribolium has a CNC structure very similar to that of T. molitor , in which the CNC has been most studied. Tribolium is an ideal species to study CNC developmental mechanisms and their molecular basis (16, 17), having a sequenced, well annotated genome, and established means for manipulating gene expression including RNAi. We previously identified Tiptop, a transcription factor of the teashirt gene family, to be expressed in Tribolium leptophragmata (17).…”

“…Here we build on these insights to gain an understanding of the molecular mechanisms by which leptophragmata identity is specified, and provide clues of their evolutionary origins. We draw on the tissue specific transcriptomic database, BeetleAtlas, which has proven a powerful resource for guiding molecular studies in Tribolium (16, 20), and conduct functional studies using the genetic manipulation, cellular morphological and physiological methods which are established for this species. We find that the transcription factor tiptop , in combination with dachshund , are required for leptophragmata identity.…”