Water Transport and Enzyme Recycling in <i>Tenebrio molitor</i> Midgut: Insights From Transcriptomics, Proteomics, and In Vivo Inhibition Assays

The low excretory rates of secreted digestive enzymes, such as trypsins, in insect species with peritrophic membranes led to the hypothesis of ectoperitrophic countercurrent water fluxes causing enzyme recycling. The midgut water flux model of Tenebrio molitor (T. molitor) is revisited and supported by in vivo experiments. Sequences from proteins putatively involved in water transport were retrieved from the T. molitor transcriptome by Blast and analyzed using bioinformatics tools. Gene expression of selected proteins was determined in three midgut sections (anterior, AM; middle, MM; posterior, PM) by RNA-seq, and transporter proteins were verified in microvillar-membrane-enriched midgut samples by proteomics. Genes encoding three cation chloride cotransporters (CCC) and four aquaporins were expressed in the midgut. TmNaCCC2, TmPrip, and TmEglp1 showed higher expression in the front half, while TmKCC, TmNKCC1, TmDrip, and TmEglp2 were more highly expressed in the back half. However, only TmNaCCC2 was found by proteomics. Midgut water fluxes were quantified by feeding T. molitor larvae with nonabsorbable dye and measuring its concentration along the midgut. The results suggest water absorption in AM and secretion in MM and PM, potentially caused by TmNaCCC2 and TmPrip in AM, and TmKCC and TmDrip in PM, whereas MM serves as a transition region. Larvae fed on furosemide, an NKCC and KCC inhibitor, showed altered midgut water fluxes, resulting in higher trypsin excretion into the hindgut, thus reinforcing the hypothesis of a countercurrent water flux generated by CCCs powering enzyme recycling in insect midguts.