Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

中文翻译：

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家蝇中肠通量和消化酶的循环利用：一种分子方法。

据报导，昆虫的肠中水逆流通量可促进排泄前的酶回收，通常称为酶循环。到目前为止，还没有单一的，非常不完整的尝试将转运蛋白和通道与逆流磁通联系起来。在这项工作中，根据沿中肠摄取的和不可吸收的染料的浓度推断家蝇中肠的水通量，其中前中肠分为两部分（A1，A2），中间一人（M），后中肠在四个（P1，P2，P3和P4）中，这导致发现其他分泌和吸收位点。水在A1和A2中分泌并在中肠（M）处吸收，而在后中肠中，水在P2处吸收，并在其他部位（主要在P4）处分泌。因此，形成了从P4到P2的逆流磁通。为了揭示已知水转运体Na +：K +：2Cl-（NKCC）和K +：Cl-（KCC）以及水通道水通道蛋白在水通量中的参与，通过一式三份样品的RNA-seq分析评估了它们的表达中肠的七个部分。MdNKCC1在A1中表达，MdNKCC2在M1和P2中表达，MdKCC在中部和最后部区域表达，因此显然分别参与分泌，吸收和两者。MdNKCC2，MdKCC和水通道蛋白MdDRIP1和2被纯化的微绒毛膜的蛋白质组学确认为顶端。通过观察速尿抑制剂的作用，测试了NKCC和KCC在中肠水通量中的作用。