Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia

https://doi.org/10.1016/j.cbpa.2019.110588Get rights and content

Highlights

  • Brilliant blue FCF has been used as a marker of Drosophila barrier failure.

  • Cold exposure can induce a Smurf phenotype in Drosophila.

  • Flies can revert back to a non-Smurf phenotype if given a recovery period.

  • Drosophila can actively secrete Brilliant blue FCF.

  • The Smurf phenotype could be caused by either transport or barrier failure, or both.

Abstract

Under conditions of stress, many animals suffer from epithelial barrier disruption that can cause molecules to leak down their concentration gradients, potentially causing a loss of organismal homeostasis, further injury or death. Drosophila is a common insect model, used to study barrier disruption related to aging, traumatic injury, or environmental stress. Net leak of a non-toxic dye (Brilliant blue FCF) from the gut lumen to the hemolymph is often used to identify barrier failure under these conditions, but Drosophila are capable of actively transporting structurally-similar compounds. Here, we examined whether cold stress (like other stresses) causes Brilliant blue FCF (BB-FCF) to appear in the hemolymph of flies fed the dye, and if so whether Drosophila are capable of clearing this dye from their body following chilling. Using in situ midgut leak and transport assays as well as Ramsay assays of Malpighian tubule transport, we tested whether these ionoregulatory epithelia can actively transport BB-FCF. In doing so, we found that the Drosophila midgut and Malpighian tubules can mobilize BB-FCF via an active transcellular pathway, suggesting that elevated concentrations of the dye in the hemolymph may occur from increased paracellular permeability, reduced transcellular clearance, or both.

Summary statement

Drosophila are able to actively secrete Brilliant blue FCF, a commonly used marker of barrier dysfunction.

Introduction

Epithelial barriers are an important constituent of all metazoan life on earth and are critical in the maintenance of homeostasis. In the gut, epithelial barriers play a major role in regulating rates of nutrient absorption; ion and water secretion and absorption; the clearance of wastes (Salvo Romero et al., 2015); and protect organisms from potential threats such as infection and dehydration (Presland and Jurevic, 2002).

Drosophila are a common model of digestive and renal physiology (Dow and Romero, 2010; Buchon et al., 2013; Miguel-Aliaga et al., 2018). The gut of Drosophila is divided into three distinct regions; the foregut, midgut and hindgut. The foregut primarily functions in food storage and regulates the passage of food into the midgut. The insect midgut is largely analogous to the small intestine in humans, and is the primary site of food digestion, and nutrient absorption (Buchon et al., 2013). From the midgut, digested material enters the hindgut, which functions in water resorption prior to defecation. The Malpighian tubules are blind-ended tubes that branch from the midgut-hindgut junction, and are a single cell layer thick (Demerec, 1950) The Malpighian tubules are responsible for the production of primary urine containing water, ions, toxins, and waste products (Dow, 2009; Buchon et al., 2013). Drosophila have four Malpighian tubules; two anterior and two posterior, which all function in ion and water transport (Denholm and Skaer, 2009; Chintapalli et al., 2012).

In addition to its critical role in facilitating our understanding of gut and renal physiology, Drosophila are also used to probe the roles of epithelial barriers in aging and disease (Dow et al., 1994; Rera et al., 2012; Rodan et al., 2012; He and Jasper, 2014; Clark et al., 2015; Dambroise et al., 2016). Advanced age, infections and thermal stress, for example, are all known to cause a loss or decrease in epithelial barrier function (Jasper, 2015; MacMillan et al., 2017), and this barrier dysfunction is thought to contribute to aging phenotypes and/or stress-induced injury or death (Katzenberger et al., 2015; Cerqueira César Machado and Pinheiro da Silva, 2016). In most of these studies, barrier dysfunction in fruit flies is identified and quantified using the “Smurf assay” (Rera et al., 2011; Martins et al., 2018). In this assay, flies are fed a food containing a high concentration of a non-toxic organic dye called Brilliant blue FCF (BB-FCF). Flies that are healthy appear to retain the dye in their gut lumen, but those that have some form of intestinal epithelial dysfunction turn blue (Rera et al., 2011). It is presumed that the presence of these Smurf phenotypes is a direct result of BB-FCF leaking down its concentration gradient (i.e. passively diffusing) from the gut lumen into the hemolymph because of barrier disruption, which will eventually make the fly appear blue (Rera et al., 2011).

When exposed to cold temperatures, D. melanogaster become less capable at regulating hemolymph osmolarity and ion balance, and as a result, hyperkalemia causes cell depolarization and cell death (MacMillan et al., 2015a). This inability to maintain ion and water balance in the cold has been attributed, in part, to a susceptibility of the renal system to chilling (Overgaard and MacMillan, 2017). Specifically, low temperatures suppress ionomotive enzyme activity in the Malpighian tubules and gut epithelia, such that a net leak of Na+ and water from the hemolymph to the gut causes a toxic increase in concentration of K+ in the hemolymph (Overgaard and MacMillan, 2017). This cascade of issues can be largely prevented in Drosophila given a period of cold acclimation (MacMillan et al., 2015b; Yerushalmi et al., 2018). When ion balance is disturbed, recovery of K+ balance (and thus cell membrane potential) is thought to be particularly important to the recovery of neuromuscular function following rewarming (MacMillan et al., 2012; Andersen and Overgaard, 2019). The ability of insects to recover can be quantified by measuring the time required for an insect to stand following chilling (termed chill coma recovery time; CCRT) (Macdonald et al., 2004; Findsen et al., 2014). Like other stressful conditions, cold exposures also cause gut barrier dysfunction (MacMillan et al., 2017), but this cold-induced barrier failure was described using another common paracellular probe (dextran) and has previously not been investigated using BB-FCF.

Some solutes are able to pass through the epithelial cells of an insect gut, while others can't (Huang et al., 2015). Large polar organic molecules (like BB-FCF or dextran) cannot easily pass through cell membranes, but there are two primary routes by which they could potentially cross epithelial barriers; transcellular and paracellular transport. Transcellular transport occurs across cell membranes via active (i.e. energy-consuming) or passive routes (O'Donnell and Maddrell, 1983). Many complex organic compounds have been shown to be actively (e.g. para-aminohippuric acid, tetraethylammonium) (Linton and O'Donnell, 2000; Rheault and O'Donnell, 2004), or passively (e.g. urea, sucrose) (Maddrell and Gardiner, 1974) transported across Drosophila epithelia. Many inorganic ions and compounds are actively transported at high rates by the gut and renal epithelia of insects (e.g. phosphate, Mg2+) (Maddrell and O'Donnell, 1992). Passive diffusion of a molecule down its concentration gradient can occur in the transcellular manner (e.g. through suitable channels) provided the molecule has a low molecular weight and is soluble in the hemolymph. By contrast, primary active transport occurs transcellularly through dedicated transporters and secondary active transport can mobilize molecules up or down a concentration gradient by coupling concentration gradients through exchangers (Maddrell and O'Donnell, 1992; Rheault and O'Donnell, 2004).

Paracellular transport is defined as the transport of molecules/solutes across the epithelial cell layer, specifically between the cells via the intercellular spaces (O'Donnell and Maddrell, 1983). In insects, paracellular permeability is regulated by the presence of septate junctions (SJ), protein complexes analogous to vertebrate tight junctions that play an important role in selectivity of passage (O'Donnell and Maddrell, 1983; Jonusaite et al., 2017). SJs act as permeability barriers to the free diffusion of molecules, and thereby control the rate at which molecules can leak down their concentration gradient from one side of an epithelium to the other (Banerjee et al., 2006; Ganot et al., 2014; Jonusaite et al., 2016). There are two types of these junctions in insects, pleated and smooth SJs; while pleated SJs are typically found in the salivary glands, epidermis and the photoreceptors of the flies, while smooth SJs are found in the midgut and Malpighian tubules (Hall and Ward, 2016; Jonusaite et al., 2017).

The mode of transport of BB-FCF through the Drosophila gut epithelia is unknown, and despite its use in understanding epithelial health and disease, whether or not flies can actively transport this dye remains untested. Importantly, other organic compounds can be actively transported by the Drosophila midgut and Malpighian tubules, including TEA, Texas red and methotrexate (Rheault and O'Donnell, 2004; O'Donnell and Leader, 2006; O'Donnell, 2009). Texas red (625.12 g mol−1), for example, is of a similar size to brilliant blue (792.85 g mol−1), and is transported across the Malpighian tubule epithelia via the action of a sodium-independent active transport mechanism (Leader and O'Donnell, 2005). This active transport has previously been confirmed by experimentally saturating the putative transporter with high concentrations of Texas red (Leader and O'Donnell, 2005).

In the present study, we hypothesized that 1) Like other stressors, cold stress can cause the Smurf phenotype in Drosophila, but that this outcome could be due to either barrier failure and/or a reduced capacity for active transport in the cold because 2) the gut and Malpighian tubule epithelia of Drosophila are innately capable of actively secreting BB-FCF. We used dye leak and clearance assays using whole animals and isolated organs to test whether and how flies transport the dye and found that cold stress does indeed cause the Smurf phenotype in Drosophila and that Smurf flies are somewhat more likely to die from cold stress. We argue that Smurf assays, however, cannot distinguish between a decrease of dye transport rates and a failure to maintain epithelial barriers under conditions of stress, as the midgut and tubules are continuously clearing the dye from the hemolymph via active transcellular transport.

Section snippets

Animal husbandry

The line of Drosophila melanogaster used in this study were derived from isofemale lines established from collections from London, Ontario and Niagara on the Lake, Ontario in 2007 (Marshall and Sinclair, 2010). D. melanogaster were reared in 200 mL bottles that contained ~50 mL of Bloomington Drosophila medium (a cornmeal, corn syrup, agar and yeast-based diet). FD&C Blue dye #1 (brilliant blue FCF, BB-FCF) was a generous gift from Calico Food Ingredients Ltd. (Kingston, ON). The flies used in

Low temperature survival and chill coma recovery

Increased cold exposure times increased the likelihood of a fly becoming a Smurf, and decreased the probability of survival following chilling. We found a significant linear relationship between the duration of exposure to 0 °C and the proportion of the flies that turned blue (i.e. became Smurfs; P = .049; Fig. 1A). Longer exposures to 0 °C also resulted in lower rates of survival Ptime < 0.001; Fig. 1B), and flies scored as Smurfs were slightly, but significantly, more likely to die from a

Discussion

Here, we demonstrate for the first time that cold stress (like advanced age or traumatic injury) causes the Smurf phenotype in Drosophila, and that the tendency to turn blue is related to cold tolerance. We also show, however, that Drosophila appears to be able to transport BB-FCF, which complicates its use as a marker of barrier disruption.

Like other stresses, cold exposure caused flies to adopt a Smurf phenotype (turn blue), and longer cold stresses increased the proportion of Smurf flies (

Data accessibility

All data is provided as a supplementary file.

Author contributions

All authors contributed to the conception and design of the study, D.L. H.P., and H.M. conducted the experiments. D.L. and H.M. analyzed the data, D.L. drafted the manuscript, and all authors edited the manuscript.

Funding

This research was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grants to both H.M. (grant RGPIN-2018-05322) and A.D. (grant RGPIN-2018-05841).

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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