Midgut pH Research Articles

α-Amylase Activities in Larval Midgut Extracts from Four Species of Lepidoptera (Tortricidae and Gelechiidae): Response to pH and to Inhibitors from Wheat, Barley, Kidney Bean, and Streptomyces

Levels of α-amylase activity between 0.6 and 5.0 µmoles maltose minute-1 gram insect-1 occurred in larval midgut extracts from 4 species of lepidopterous pests-light brown apple moth, Epiphyas postvittana (Walker), Planotortrix octo (Dugdale), and Ctenopseustis Obliquana (Walker) are pests of apple leaves and fruit, and potato tuber moth, Phthorimaea Operculella (Zeller), is a pest of potato tubers and foliage. Activity response curves described the optima for activity at a highly alkaline pH range (pH 10.5 in leafrollers and pH 9.5 in potato tuber moth). This result is consistent with the known high midgut pH in other species of Lepidoptera and with the pH optima of their digestive proteases. The 3 wheat derived α-amylase inhibitors inhibited enzyme activities of all 4 species of Lepidoptera, but the other 3 (barley tetramer, Streptomyces, and kidney bean inhibitors) had no effect. Wheat dimeric inhibitor (30 µM) completely inhibited α-amylase activity in potato tuber moth midgut extracts.

Potential influence of midgut pH and redox potential on protein utilization in insect herbivores

Early insect physiologists recognized the importance of gut physicochemistry, primarily pH, redox potential, and ionic strength, on digestive processes, but studies to date have been primarily restricted to keratin- and wood-feeding insects. Recent investigations show that herbivorous insects have a broad range of gut redox conditions, with pHs ranging from 6.0 to 11.8 and measured redox potentials from −200 to +240 mV. The redox state of the gut is largely dependent on pH, which is well regulated, and the redox activity of ingested material, including plant chemicals, at that pH. Inter- and intraspecific variation in midgut redox conditions appears to be substantial enough to affect digestion via effects on the structure and function of dietary proteins and proteolytic enzymes. The impact of reducing conditions on proteins probably depends on characteristics such as tertiary structure and the number and arrangement of disulfide linkages. In addition to the effects of reducing conditions in dietary proteins, there can be effects on the activities of digestive enzymes, depending on their structure and the nature of their catalytic site. We speculate that phylogenetic and environmental determinants of gut physicochemistry may place constraints on the efficacy of different digestive processes, and may thus influence the evolution of digestive strategies in insects. © 1996 Wiley-Liss, Inc.

PH buffering in Musca domestica midguts

The M. domestica midgut displays three morphological regions with the following luminal pH values: anterior, 6.1; middle, 3.1; posterior, 6.8. Looking for enzymes that might be related to the acidification of middle midguts or to the neutralization of luminal contents in the anterior and posterior midguts, M. domestica larvae were placed on layers of 10% starch gels containing 0.1% Congo Red or 0.1% lacmoid and one of the following compounds: acetazolamide, vanadate, KSCN, NaF, ouabain, calcium acetate, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, or amiloride. None of these compounds induced alteration in anterior or posterior midgut pH, whereas acetazolamide, vanadate, NaF, and ouabain affected luminal pH in middle midgut. Ammonia and phosphate predominate in anterior and posterior midguts, chloride concentrates in middle midgut, and bicarbonate occurs in low concentration along the midgut lumen. Ouabain and vanadate cause a relative decrease of chloride in the middle midgut and an increase of ammonia mainly in the posterior midgut. The results suggest that chloride may follow the protons translocated into middle midgut contents by a type-P ATPase, similar to that found in vertebrate stomachs, which is inhibited by high intracellular concentration of Na +. Ammonia probably neutralizes the contents of the anterior and mainly posterior midguts and is secreted by mechanism probably involving an apical Na + K + -ATPase .

Physiological and Nutritional Effects of a Fluorescent Brightener on Nuclear Polyhedrosis Virus-Infected Lymantria dispar (L.) Larvae (Lepidoptera: Lymantriidae)

Abstract Recent studies with gypsy moths ( Lymantria dispar L.) show that the fluorescent brightener, Tinopal LPW, acts synergistically with the L. dispar nuclear polyhedrosis virus (LdMNPV), lowering the 50% lethality concentration of the virus (LC 50 ) by 1000-fold. As a continuation of this work, we investigated the effects of ingestion of LdMNPV/Tinopal on larval midgut pH, hemolymph pH, and nutritional indices. Larval feeding for 48 h on diet treated with either LdMNPV or Tinopal did not affect midgut luminal pH, which averaged 10.3. In contrast, after 48 h of larval feeding on LdMNPV/ Tinopal diet, midgut pH averaged 8.5, and frass production, weight gain, and larval nutritional indices were significantly reduced. These effects appeared to be irreversible after the first 24 h of feeding ad libitum on LdMNPV/Tinopal diet, and the reduction in midgut pH was not due to feeding cessation per se. No significant effects on hemolymph pH were detected with any treatment. No treatment effects were observed when SITS, a related fluorescent brightener, was substituted for Tinopal. The severe biological perturbations resulting from LdMNPV/Tinopal consumption are interpreted in light of current understanding of larval lepidopteran midgut physiology.

The effect of maturation time on the subsequent in vitro development of bovine oocytes

The M. domestica midgut displays three morphological regions with the following luminal pH values: anterior, 6.1; middle, 3.1; posterior, 6.8. Looking for enzymes that might be related to the acidification of middle midguts or to the neutralization of luminal contents in the anterior and posterior midguts, M. domestica larvae were placed on layers of 10% starch gels containing 0.1% Congo Red or 0.1% lacmoid and one of the following compounds: acetazolamide, vanadate, KSCN, NaF, ouabain, calcium acetate, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, or amiloride. None of these compounds induced alteration in anterior or posterior midgut pH, whereas acetazolamide, vanadate, NaF, and ouabain affected luminal pH in middle midgut. Ammonia and phosphate predominate in anterior and posterior midguts, chloride concentrates in middle midgut, and bicarbonate occurs in low concentration along the midgut lumen. Ouabain and vanadate cause a relative decrease of chloride in the middle midgut and an increase of ammonia mainly in the posterior midgut. The results suggest that chloride may follow the protons translocated into middle midgut contents by a type-P ATPase, similar to that found in vertebrate stomachs, which is inhibited by high intracellular concentration of Na+. Ammonia probably neutralizes the contents of the anterior and mainly posterior midguts and is secreted by mechanism probably involving an apical Na+K+-ATPase.

Avoidance of antinutritive plant defense: Role of midgut pH in Colorado potato beetle

The fate of the tomato foliar phenolic, chlorogenic acid, in the digestive systems of Colorado potato beetleLeptinotarsa decemlineata (Coleoptera: Chrysomelidae) andHelicoverpa tea (Lepidoptera: Noctuidae) is compared. In larvalH. zea and other lepidopteran species previously examined, approximately 35-50% of the ingested chlorogenic acid was oxidized in the digestive system by foliar phenolic oxidases (i.e., polyphenol oxidase and peroxidase) from the tomato plant. The oxidized form of chlorogenic acid, chlorogenoquinone, is a potent alkylator of dietary protein and can exert a strong antinutritive effect upon larvae through chemical degradation of essential amino acids. In contrast, inL. decemlineata less than 4% of the ingested dose of chlorogenic acid was bound to protein. In vitro experiments to determine the influence of pH on covalent binding of chlorogenic acid to protein showed that 30-45% less chlorogenic acid bound to protein at pHs representative of the beetle midgut (pH 5.5-6.5) than at a pH representing the lepidopteran midgut (pH 8.5). At an acidic pH, considerably more of the alkylatable functional groups of amino acids (-NH2, -SH) are in the nonreactive, protonated state. Hence, polyphenol oxidases are unlikely to have significant antinutritive effects against the Colorado potato beetle and may not be a useful biochemical source of resistance against this insect. The influence of feeding by larval Colorado potato beetle on foliar polyphenol oxidase activity in tomato foliage and its possible significance to interspecific competition is also considered.

Grasshopper (Orthoptera: Acrididae) Midgut pH in Relation to Development, Starvation, and Species: Implications of a New, Fast-Freezing Methodology

Laboratory-reared and field-collected grasshoppers were assayed for midgut pH following live extractions and three freezing techniques. Grasshoppers were frozen whole using liquid propane and liquid nitrogen (-196°C) or by placement in a freezer (-20°C). Midgut pH of laboratory-reared Melanoplus differentialis (Thomas) changed little with starvation or development from third instar through adult. Midgut pH of seven field-collected species ranged from 6.41 to 6.68, with males generally having a higher pH than females. Across all tested developmental stages of M. differentialis and feeding regimes (starved and fed), midgut pH of grasshoppers frozen instantly in liquid propane was significantly higher than those of grasshoppers frozen slowly. Freezing of adult M. sanguinipes (F.) in liquid propane or nitrogen led to lower pH mean measurements and smaller variances than with live extractions. Thus, pH measurements are highest with live extractions, followed by fast-freezing, and lowest with slow freezing. The new methodology provides the most accurate and precise measurement of normal physiological conditions in the midgut to date, which is critical to understanding pathogen host ranges and developing production systems for microsporidian and other ingested pathogens of grasshoppers.

The effect of diet on gypsy moth ( Lymantria dispar) larval midgut pH, and its relationship with larval susceptibility to a baculovirus

The midgut pH of gypsy moth ( Lymantria dispar) larvae was significantly lower when larvae were fed acidic folicage. At 30 min after the start of a feeding bout, larvae feeding on bigtooth aspen (foliage pH = 6.04) had average midgut pH levels of 10.16, while larvae feeding on red oak foliage (foliage pH = 3.84) had average midgut pH levels of 9.36. Midgut pH was also significantly reduced when larvae were fed acidic artificial diets. At 30 min, average midgut pH levels were 9.80 and 9.42 for larvae fed diets buffered at pH 7 and 4, respectively. Larval susceptibility to the gypsy moth nuclear polyhedrosis virus was also significantly reduced when larvae consumed virus on highly acidic foliage and acidic artificial diets. Diet pH may affect virus activity by changing midgut pH and altering the rate and location of virion release in the midgut lumen.

Digestion and excretion of nitrogen and carbohydrate by the cranefly larva Tipula paludosa (Diptera: Tipulidae)

The nitrogenous and carbohydrate components of ryegrass and faeces from larvae of Tipula paludosa Meigen, fed on ryegrass ( Lolium perenne L.), were compared. Proteins in ryegrass were efficiently digested and uric acid was the major nitrogenous excretory product. The alkaline midgut (pH 9.1) was considered to enhance the digestibility of hemicellulose, by removing inhibitory acetyl groups, and of cellulose by altering its crystallinity. T. paludosa larvae assimilated 50% of ryegrass cellulose, and 50% of an isolated 14C-labelled cellulose, whereas 86% of hemicellulose was digested.

Hostplant, larval age, and feeding behavior influence midgut pH in the gypsy moth (Lymantria dispar).

The midgut pH of late instar gypsy moth (Lymantria dispar L.) larvae is strongly alkaline, and varies with diet, larval stadium, and time since feeding. Midgut pH rises with time since feeding, and does so more quickly, reaching greater maximum values, on some diets than others. Leaf tissues of 23 tree species resist increases in alkalinity differentially; this trait and differing initial leaf pH may explain the impact of diet on gut pH. Third instar larvae may have gut conditions favorable for tannin-protein binding shortly after ingesting certain foods, but with time midgut alkalinity becomes great enough to dissociate tannin-protein complexes. Older instars rarely exhibit gut pHs low enough to permit tannin activity. Alkaline gut conditions may explain the gypsy moth's ability to feed on many tanniniferous plant species, especially in later instars. Consequences for pathogen effectiveness are discussed.

Midgut pH in different instars of three Aedes mosquito species and the relation between pH and susceptibility of larvae to a nuclear polyhedrosis virus

Midgut pH values were determined for second, third, and fourth instar larvae of Aedes epactius, A. atropalpus, and A. scutellaris. Larvae were fed mixtures of pH indicators and kaolin clay and observed with a stereoscope to determine midgut pH. No significant difference was found in midgut pH values among the three species when the same instar was compared. However, significant differences were found in the length of the high (9.0 to 10.0) pH region when comparing the three instars for each species. Increasing age of treated A. epactius larvae caused a decrease in susceptibility to a nuclear polyhedrosis virus—from 56% infection for 12-hr-old larvae declining to 8% infection for 72-hr larvae. The increased length of the high pH region may relate to decreasing susceptibility of A. epactius to a nuclear polyhedrosis virus as the larvae age.

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH 4) 2SO 4, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia. Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0. The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH 4) 2SO 4 and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.

Susceptibility of Japanese beetle larvae to Bacillus thuringiensis: Associated effects of diapause, midgut pH, and milky disease

Susceptibility of Japanese beetle larvae to Bacillus thuringiensis: Associated effects of diapause, midgut pH, and milky disease

Susceptibility of Populations of Indianmeal Moth and Almond Moth to Bacillus thuringiensis123

Concentration-mortality studies with several populations of the Indianmeal moth, Plodia interpunctella (Hubner), and the almond moth, Ephestia cautella (Walker), were conducted with Bacillus thuringiensis subsp. kurstaki Berliner incorporated into a ground wheat diet. Indianmeal moth and almond moth populations differed as much as 6- and 10-fold, respectively, in responses to B. thuringiensis . The differences in both cases appeared to be related, in part, to population vigor. Midgut pH did not relate to population susceptibility in either species.

Microbial synergists pathogenic to Lymantria dispar: Chitinolytic and fermentative bacterial interactions

A chitinolytic bacterium isolated from a healthy gypsy moth, Lymantria dispar, larva was shown to be pathogenic to larvae when administered per os after growth on chitin broth. However, the chitin hydrolase produced by this isolate had a pH optimum for activity of 5.5 and the high alkalinity in the mesenteron of L. dispar larvae severely limited chitinolysis of the midgut lining. Fermentative, nonpathogenic, acid-producing bacteria isolated from healthy gypsy moth larvae effectively lowered larval mesenteron pH when administered per os and the combination of fermentative isolates with a crude culture aliquot of the chitinolytic strain produced a synergistic increase in mortality over either dose administered by itself. Increased mortality was also observed for most fermentative strains when they were combined with crude supernatants of centrifuged cultures of the chitinolytic strain, although these combinations proved less effective than when fermenters were added to the whole-culture aliquots of the chitinolytic strain. In vitro studies showed that other bacteria isolated from environments foreign to that of the gypsy moth could ferment carbohydrates with acid production at an alkaline pH; however, in vivo studies demonstrated that these bacteria were incapable both of poising larval midgut pH and of enhancing mortality when added to chitinolytic bacteria.

Some Factors Affecting the Pathogenicity of Bacillus thuringiensis Berliner Against Blackflies 1

Several factors that might affect the efficacy of Bacillus thuringiensis against Simulium vittatum larvae were investigated in the laboratory utilizing a conical-flushing rearing unit and the jar method for bioassay. S. vittatum and S. argus were considerably more susceptible to B. thuringiensis than the other species tested. There was a strong positive correlation between temperature and mortality (r = 0.97). The range of mortality for 4°–24°C was 23–95%. The midgut pH ranged from 8.2–11.4, sufficiently high for θ-endotoxin activity. When spore and crystal fractions were bioassayed separately, only the crystal fraction produced significant mortality. No additive action of the spore and crystal was apparent, activity was solely attributed to the parasporal crystalline inclusion bodies. Spore preparations autoclaved and not autoclaved, containing the β -exotoxin produced a low level of mortality at 10 ppm for 24 h. Spore suspensions of B. thuringiensis stored at 2° and 19°C remained active for 167 and 28 days, respectively, after which a sharp decline in activity was observed.

In vivo germination and host specificity of Nosema algerae in mosquitoes

Of five species of mosquitoes tested, Anopheles stephensi and A. albimanus were the most susceptible, A. quadrimaculatus and Culex pipiens were relatively unsusceptible and A. atroparvus was almost completely refractory to Nosema algerae. Spore germination percentages differed in the larval midguts of the five species of mosquito but not in the order which would explain the susceptibility differences. Neither midgut pH difference nor differences in passage rate through the midgut were sufficient to explain the observed differences in spore germination percentages.