Larvae of , the yellow fever vector, inhabit a variety of aquatic habitats ranging from freshwater to brackish water. This study focuses on the gastric caecum of the larvae, an organ that has not been widely studied. We provide the first measurements of H, K and Na fluxes at the distal and proximal gastric caecum, and have shown that they differ in the two regions, consistent with previously reported regionalization of ion transporters. Moreover, we have shown that the regionalization of vacuolar H-ATPase and Na/K-ATPase is altered when larvae are reared in brackish water (30% seawater) relative to freshwater. Measurements of luminal Na and K concentrations also show a 5-fold increase in Na/K ratio in the caecal lumen in larvae reared in brackish water relative to freshwater, whereas transepithelial potential and luminal pH were unchanged. Calculated electrochemical potentials reveal changes in the active accumulation of Na and K in the lumen of the gastric caecum of freshwater versus brackish water larvae. Together with the results of previous studies of the larval midgut, our results show that the caecum is functionally distinct from the adjacent anterior midgut, and may play an important role in osmoregulation as well as uptake of nutrients.
Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.
We report measurements of ion transport across the gastric caecum of larvae of , a vector of yellow fever that inhabits a variety of aquatic habitats ranging from freshwater to brackish water. We provide the first measurements of the effect of 5-hydroxytryptamine (5-HT) on transepithelial potential (TEP), luminal ion concentrations and electrochemical potentials, as well as basolateral membrane potential and H, Na and K fluxes. TEP, basolateral membrane potential, and H, K and Na fluxes across the gastric caeca declined within 3-6 min after isolation of the entire midgut from the larva. 5-HT restored both the TEP and active accumulation of H, K and Na in the lumen. Additionally, 5-HT restored H, K and Na fluxes across the distal caecum of freshwater larvae, and restored H fluxes across the distal caecum of brackish water larvae. There was no effect of 5-HT on ion fluxes across the proximal caecum. We have also shown that 5-HT restores the basolateral membrane potential in cells of the distal, but not proximal, caecum. Effects of 5-HT on TEP and basolateral membrane potential were mimicked by application of cAMP but not by a phorbol ester. We provide a working model which proposes that 5-HT and cAMP stimulate the vacuolar H-ATPase of the distal caecum. Our results provide evidence that the gastric caecum is functionally distinct from the adjacent anterior midgut and we discuss possible roles of the gastric caecum in osmoregulation. We also describe similarities in the arrangement of ion transporters in the caecum with those of the Malpighian tubules.
Alcohol use disorder (AUD) is characterized by loss of control in limiting alcohol intake. This may involve intermittent periods of abstinence followed by alcohol seeking and, consequently, relapse. However, little is understood of the molecular mechanisms underlying the impact of alcohol deprivation on behavior. Using a new Drosophila melanogaster repeated intermittent alcohol exposure model, we sought to identify how ethanol deprivation alters spontaneous behavior, determine the associated neural structures, and reveal correlated changes in brain gene expression. We found that repeated intermittent ethanol-odor exposures followed by ethanol-deprivation dynamically induces behaviors associated with a negative affect state. Although behavioral states broadly mapped to many brain regions, persistent changes in social behaviors mapped to the mushroom body and surrounding neuropil. This occurred concurrently with changes in expression of genes associated with sensory responses, neural plasticity, and immunity. Like social behaviors, immune response genes were upregulated following three-day repeated intermittent ethanol-odor exposures and persisted with one or two days of ethanol-deprivation, suggesting an enduring change in molecular function. Our study provides a framework for identifying how ethanol deprivation alters behavior with correlated underlying circuit and molecular changes.
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