Na+/H+-exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown.Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH ∼9) and low [Na+] ( ̴120 mM) in their midgut fluids, compared to the ionic composition of the surrounding sea water. We pharmacologically investigated the role of Na+/H+-exchangers in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application, via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using vivo morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with qPCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8 that potentially contribute to the regulation of [Na+] and pH in midgut fluids.This work provides new insights to ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features to insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.

Glucose-mediated expansion of a gut commensal bacterium promotes Plasmodium infection through alkalizing mosquito midgut

SUMMARYDietary sugar is the major energy source for mosquitoes, but its influence on mosquitoes’ capability to transmit malaria parasite remains unclear. Here we show that Plasmodium berghei infection changes global metabolism of Anopheles stephensi with the most significant impact on glucose metabolism. Supplementation of glucose or trehalose (the main hemolymph sugar) to mosquito increases Plasmodium infection by alkalizing the mosquito midgut. The glucose/trehalose diets promote rapid growth of a commensal bacterium, Asaia bogorensis, which remodels glucose metabolism and consequently increases midgut pH. The pH increase in turn promotes Plasmodium gametogenesis. We also demonstrate the sugar composition from different natural plants influences A. bogorensis growth and Plasmodium infection is associated with their capability to expand A. bogorensis. Altogether, our results demonstrate that dietary glucose is an important factor that determines mosquito’s competency to transmit Plasmodium and further highlight a key role for mosquito-microbiota metabolic interactions in regulating development of malaria parasite.

Detoxification of host plant phenolic aglycones by the spruce budworm

This study examines the post-ingestive fate of two host-plant derived small-molecule phenolics (the acetophenones piceol and pungenol) that have previously been shown to be toxic to the outbreaking forest pest, spruce budworm (Choristoneura fumiferana). We test first whether these compounds are transformed during passage through the midgut, and second whether the budworm upregulates activity of the detoxification enzyme glutathione-s-transferase (GST) in response to feeding on these compounds. Insects were reared on either foliage or artificial diet to the fourth instar, when they were transferred individually to one of two treatment diets, either control or phenolic-laced, for approximately 10 days, after which midguts were dissected out and used for Bradford soluble protein and GST enzyme activity analysis. Frass was collected and subjected to HPLC-DAD-MS. HPLC showed that the acetophenones do not autoxidize under midgut pH conditions, but that glucose- and glutathione-conjugates are present in the frass of insects fed the phenolic-laced diet. GST enzyme activity increases in insects fed the phenolic-laced diet, in both neutral pH and alkaline assays. These data show that the spruce budwom exhibits counter-adaptations to plant phenolics similar to those seen in angiosperm feeders, upregulating an important detoxifying enzyme (GST) and partially conjugating these acetophenones prior to elimination, but that these counter-measures are not totally effective at mitigating toxic effects of the ingested compounds in the context of our artifical-diet based laboratory experiment.

Controlled-release ofBacillus thurigiensisformulations encapsulated in light-resistant colloidosomal microcapsules for the management of lepidopteran pests ofBrassicacrops

Bacillus thuringiensis(B. t.) based formulations have been widely used to control lepidopteran pests in agriculture and forestry. One of their weaknesses is their short residual activity when sprayed in the field. Using Pickering emulsions, mixtures of spores and crystals from threeB. t.serovars were successfully encapsulated in colloïdosomal microparticles (50 μm) using innocuous chemicals (acrylic particles, sunflower oil, iron oxide nanoparticles, ethanol and water). A pH trigger mechanism was incorporated within the particles so thatB. t.release occurred only at pH > 8.5 which corresponds to the midgut pH of the target pests. Laboratory assays performed onTrichoplusia ni(T. ni) larvae demonstrated that the microencapsulation process did not impairB. t.bioactivity. The best formulations were field-tested on three key lepidopteran pests that attackBrassicacrops, i.e., the imported cabbageworm, the cabbage looper and the diamondback moth. After 12 days, the mean number of larvae was significantly lower in microencapsulated formulations than in a commercialB. t.formulation, and the effect of microencapsulated formulations was comparable to a chemical pesticide (lambda-cyhalothrin). Therefore, colloïdosomal microcapsule formulations successfully extend the bioactivity ofB. t.for the management of lepidopteran pests ofBrassicacrops.

Digestive tract and leaf processing capacity of the stream invertebrate Tipula lateralis

We investigated the digestive potential of the stream detritivore Tipula lateralis Meigen, 1804 as a tool to understand and predict their ability to adapt to environmental changes, such as the nature of leaf resources reaching streams. The structure of larval digestive tracts and the presence of bacteria were investigated by scanning and transmission electron microscopy. Gut sections were investigated with respect to pH, microbial counts, and ability to digest protein and carbohydrates of unconditioned leaves of Alnus glutinosa (L.) Gaertn. and Eucalyptus globulus Labill. Alkaline conditions prevailed throughout the gut, with the highest values in the middle midgut (pH up to 10.8). An abundant microflora was closely associated with the caecum (~34 × 104 colony forming units (cfu)) and hindgut (~50 × 104 cfu), whereas the foregut and midgut seemed to be almost devoided of permanent bacteria (~0.45 × 104 to ~2.1 × 104 cfu). Digestion of T. lateralis seems to be optimized by compartmentalization: proteinase activity was limited to the midgut (0.017 ± 0.0054 change in absorbance units), while the capacity to release reducing substances was almost completely restricted to the caecum and being higher when alder was used as a substratum (0.437 ± 0.153 g glucose·mg–1·h–1). Eucalyptus oils resulted in a 25% reduction in enzymatic capability of gut extracts. Overall, the results suggest that T. lateralis has the enzymatic capability of feeding on recalcitrant leaves, but specific components of leaves such as oils have the potential to interfere with food intake by this leaf consumer. In a more general context, the results help to assess biological changes introduced by eucalyptus plantations in non-native areas.

Borrelia burgdorferi Proteins Whose Expression Is Similarly Affected by Culture Temperature and pH

ABSTRACT Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, ofBorrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34°C but not at 24°C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of B. burgdorferi proteins, which in culture is selectively expressed under conditions of 34°C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated.

Antibody to H(+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti)

The pH profile, gross structure, ultrastructure and immunolabeling of the mosquito (Aedes aegypti) larval midgut are described as a first step in analyzing the role of plasma membrane H(+)V-ATPase in the alkalization of the gut, nutrient uptake and ionic regulation. Binding of an antibody to H(+)V-ATPase subunit E colocalizes with ‘portasomes’ (approximately 10 nm in diameter), which are thought to correspond to the V(1) part of the H(+) V-ATPase. In gastric caeca (pH 8), both antibody-binding sites and portasomes are located apically; in the anterior midgut (pH 10–11), they are located basally; and in the posterior midgut (pH approximately equal to 8) they are again located apically. The hypothesis that the energization of alkalization is mediated by an H(+) V-ATPase is supported by the inability of larvae to maintain the high pH after 72 h in 10 (micro)M bafilomycin B1. Confirming earlier reports, the two principal epithelial cell types are designated as ‘columnar’ and ‘cuboidal’ cells. The apical plasma membranes (microvilli) of epithelial cells in the gastric caeca and basal infoldings of anterior midgut are invaded by mitochondria that lie within approximately 20 nm of the portasome-studded plasma membranes. The colocalization of V-ATPase-immunolabeling sites and portasomes to specific plasma membranes within so-called ‘mitochondria-rich’ cells of gastric caeca and anterior midgut suggests that midgut alkalization in mosquitoes is achieved by molecular mechanisms similar to those that have been described in caterpillars, even though the gross structure of the midgut and the localization of the V-ATPase are dissimilar in the two species. In caterpillars, the high alkalinity is thought to break down dietary tannins, which block nutrient absorption; it may play a similar role in plant-detritus-feeding mosquito larvae. The colocalization of immunolabeling sites and portasomes, together with the presence of long, ‘absorptive-type’ microvilli in the posterior midgut, suggest that the V-ATPase energizes nutrient uptake there.