Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach

Highlights

• The countercurrent flux of water occurs within posterior midgut.

• Symporters NKCC and KCC are expressed in midgut sections involved in water fluxes.

• Inhibition of NKCC and KCC affect midgut water fluxes.

• Inhibition of water fluxes impaired trypsin recycling increasing its excretion.

Abstract

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.

Introduction

Midgut fluid fluxes were demonstrated for the first time by Wigglesworth (1933), using dyes when studying osmoregulation in mosquito larvae. He stated that fluid is secreted by the posterior midgut and absorbed by the ceca at the anterior midgut. Treherne (1959) in his studies on amino acid absorption found that the midgut ceca of locust were the site of water absorption. Based on locust and mosquito data, Berridge (1970) proposed that digestion proceeds along the entire length of the midgut, with digestive residues accumulating in the posterior midgut and the products of digestion being directed towards the absorptive sites in the anterior midgut. The proposals of Berridge (1970) have met with wide acceptance, although at that time there were no quantitative data on midgut fluid fluxes or even a clear evidence for a secretion of fluid in the posterior midgut.

The first quantitative study of midgut fluid fluxes was done by Dow (1981), using dyes and other techniques to conclude that in starving locusts the anterior midgut ceca absorbs water coming from the Malpighian tubules, resulting in a fluid flux opposite to the food bolus movement. However, in fed locusts such countercurrent flux was not observed as a result of the saturation of the absorptive capacity of the anterior ceca by salivation.

The relationship between countercurrent fluxes and enzyme recycling to avoid excretion was first established by Terra and Ferreira (1981). They measured fecal digestive enzymes delivered at different times onto humid sand layers by the larvae of Rhynchosciara americana. After taking into account the recoveries of the enzymes from the sand layers and the stabilities of the enzymes inside the gut, excretory rates were calculated. The fact that trypsin from inside the peritrophic membrane (PM) was excreted at a low rate equal to that of the enzymes restricted to the luminal space outside PM, led them proposing that trypsin was transferred from inside PM to outside PM at the end of the posterior midgut and then directed forward by a countercurrent flux, before being excreted. Once at anterior region of posterior midgut, trypsin enters again inside PM (that is, trypsin was recycled). As expected from this recycling mechanism, trypsin is more active at the anterior region than at the posterior region of posterior midgut and this difference decreases in the presence of excess protein that also led to an increase in trypsin excretion (Terra and Ferreira, 1981). A more detailed investigation of enzyme recycling was carried out with the larva of housefly (Diptera, Cyclorrhapha, Musca domestica) (Espinoza-Fuentes et al., 1987). With dyes added to the diets, the authors calculated the volumes of water secreted and absorbed at different parts of the midgut of the housefly larva and proposed that a countercurrent flux of fluid occurs outside PM from the end of the posterior midgut to the middle midgut and that it was responsible for decreasing the excretion rate of trypsin. These findings have been extended to include Lepidoptera (Bolognesi et al., 2001, Bolognesi et al., 2008), Coleoptera (Ferreira et al., 2002; Caldeira et al., 2007), Orthoptera Ensifera (Biagio et al., 2009), and Phasmida (Monteiro et al., 2014). In contrast to locusts, all mentioned insects present countercurrent fluid fluxes in both starved and fed animals. Midgut countercurrent fluxes were also suggested by the accumulation of proteins in anterior midgut which were secreted in middle (Peterson et al., 1994) or posterior (Borhegyi et al., 1999) midgut.

Bolognesi et al., (2008) applied an algorithm to compute enzyme distribution along the midgut contents, given the water secretion and absorption sites and enzyme secretory site. The calculated distribution of enzyme activities along the posterior midgut contents of M. domestica only reflects actual experimental data if the site of water absorption was put in the anterior region of the posterior midgut, instead of in the middle midgut, indicating that something is missing in the Espinoza-Fuentes and Terra model. Besides, cyclorrhaphous larvae have a constriction region between the middle and posterior midgut that greatly reduce the lumen in the middle-posterior boundary, probably collapsing the ectoperitrophic space (Buchon et al., 2013; Richards et al., 2015). As a consequence of this constriction and midgut peristalsis, the movement of the luminal contents is forward, rather than backward. Despite the numerous papers dealing with midgut countercurrent flux of fluids and enzyme recycling, there is a lack of data on the molecular mechanisms underlying this phenomenon.

Symporters belonging to the Cation-Chloride Cotransporter family (CCC; SLC12) are expressed in insect tissues involved in ion transport and water balance as gut, anal pads and Malpighian tubules (Sun et al., 2010; Gillen et al., 2006; Piermarini et al., 2017; Rodan et al., 2012). The CCC family include Na⁺:K⁺:2Cl⁻ (NKCC) and K⁺:Cl⁻ (KCC) symporters, which are electroneutral and some of them were characterized as water symporters in mammals. In this case the ions are followed by hundreds of water molecules, allowing the symporters to function as water pumps (Zeuthen, 2010).

NKCC exists in two isoforms in mammals with different cellular localization. NKCC1 is ubiquitously expressed, mainly at basolateral membrane, whereas NKCC2 is found predominantly at apical membrane of the epithelium of the thick ascending limb of the loop of Henle of the kidney. Regardless the cellular localization (apical or basolateral) NKCCs transport ions and water into the cell using the large inwardly directed electrochemical gradient of Na⁺, whereas KCCs transport ions and water out of the cell using the outwardly directed electrochemical gradient of K⁺. The electrochemical gradient is expected to be maintained by the Na⁺/K⁺-ATPase activity, which compensates the Na⁺ influx of NKCCs and K⁺ efflux of KCCs (Zeuthen, 2010). Furthermore, the symporters are inhibited by furosemide which is largely used to test the physiological role of NKCC and KCC (Gamba, 2005; Arroyo et al., 2013; Shekarabi et al., 2017).

Insect AQPs has being classified in four families: DRIP, water-selective AQPs; PRIP, water-urea AQP; EGLP, water-urea-glycerol AQP (specific of holometabolans) (Finn et al., 2015) and BIB. BIB protein has a high sequence similarity and is phylogenetically related with aquaporins. However, BIB does not transport water. It actually functions as an ion channel (Yanochko and Yool, 2002).

The single attempt to provide a comprehensive working molecular model for midgut countercurrent flux in insects was developed only from a transcriptomic approach. According to this model, water is secreted by the posterior midgut of Tenebrio molitor with the help of KCC and absorbed back at the anterior midgut through a NKCC (Moreira et al., 2017). As transport systems among insects may vary according to the taxonomic group, the use of a transcriptomic approach combined with other supporting techniques may provide interesting results if applied to insects pertaining to different orders.

The present work was undertaken with M. domestica larvae to identify water-secreting and water- absorbing sites along the midgut and to describe the microvillar midgut transporters which might be responsible for them. With the use of a specific transporter inhibitor, the role of the identified transporters in water movements was confirmed. Finally, the role of a countercurrent flux in enzyme recycling was followed by assaying trypsin activity along the midgut and at the hindgut. The results support a model of midgut countercurrent fluxes that powers trypsin recovery avoiding excretion, a phenomenon usually referred to as enzyme recycling.