Previous studies of selective reabsorption in insect excretory system have concentrated almost exclusively on the rectum, while the role of the ileum has been assumed to be minor. The properties and control of solute and fluid transport in two segments of the hindgut, the ileum and rectum, from the desert locust (Schistocerca gregaria) have been studied and compared in vitro using everted sac and flat sheet preparations. Everted sacs of locust ileum transported fluid from the lumen side to hemocoel side over a 5 h period at near constant rates of 3.0 to 3.5 μL-h⁻¹-ileum⁻¹ and tissue volume did not change. Inhibition by azide indicated metabolic dependence of fluid transport. Fluid absorption occurred against osmotic concentration differences of up to 600 mosmol. Fluid transport was stimulated by cAMP, both nervous and glandular lobes of corpus cardiacum (CC), and fifth ventral ganglia (VG) in a dose-dependent manner. All stimulants caused ilea to absorb against larger osmotic concentration differences than unstimulated sacs. The ileal absorbate remained hyperosmotic to the luminal saline under all conditions and stimulants increased absorbate osmolality. Unstimulated fluid transport was supported at 50% of control levels by any one of Na+, K+, or CI⁻. Stimulation of fluid transport by CC or VG was dependent on CI⁻ and maximal stimulation occurred when the Na+:K+ ratio was 1:1. Cyclic AMP, CC and VG all stimulated Na+,K+ and CI⁻ absorption across everted ileal sacs. This is the first direct demonstration that Na+ reabsorption is controlled in insect excretory systems. Stimulation resulted in a decrease in absorbate HCO₃⁻ levels and pH concurrently with an increase in absorbate CI⁻ levels. Stimulation of fluid transport was associated with a 3-fold increase in transepithelial potential (hemocoel negative) suggesting stimulation of electrogenic anion (CI⁻) movement to the hemocoel. Net Na+ absorption occurs largely by electroneutral active transport. NH₄+/Na+ exchange may account for one-third of stimulated net Na+ flux. Extracts from both CC and VG stimulated fluid, K+, and CI⁻ transport across everted rectal sacs, but only a small stimulation of Na+ flux was observed which was an order of magnitude less than that observed for stimulated ilea (0.4 versus 5.1 μequiv-h⁻¹ -cm⁻²). Unlike the rectum, the ileum did not transport proline transepithelially and ileal fluid transport was not stimulated by increasing concentrations of proline in the bathing saline. Rectal fluid transport was stimulated 50% by increasing external proline concentration from 1 to 80 mM. Stimulation of rectal fluid transport by proline also occurred in the absence of Na+, K+, and CI⁻ and occurred against larger osmotic concentration differences. These results are consistent with previous reports of a high capacity transport system for proline in locust rectum. The presence of anion-stimulated ATPase and Na+,K+-ATPase in locust hindgut was also investigated. Anion-stimulated ATPase activities were observed in microsomal fractions of both rectum and ileum. Microsomal fractions from both tissues had enriched specific activities of several plasma membrane marker enzymes and decreased activities of two mitochondrial markers as compared to homogenate enzyme activities. Na+,K+-ATPase activity was 20-fold higher in the rectum than in the ileum, associated with the greater development of the basolateral membrane in the rectum. Overall the results suggest that ion and fluid reabsorption in the locust ileum is much more important in the excretory process than previously supposed. Moreover, this reabsorption was shown to be under neuroendocrine control. / Science, Faculty of / Zoology, Department of / Graduate

No description available.

The antibiotic fungal metabolite sinefungin is a potent inhibitor of S-adenosylmethionine-acceptor methyltransferases. Its effect on insect metabolism and especially on corpora allata farnesoic acid methyltransferase, which catalyzes the penultimate step of juvenile hormone biosynthesis, was investigated in Locusta migratoria. Injection of sinefungin results in a delay of imaginal molt and in suppression of ovary development. Isolated corpora allata are unable to synthesize juvenile hormone III in the presence of more than 1.0 mM sinefungin. In a cell-free system containing the S-adenosylmethionine-dependent farnesoic acid methyltransferase from corpora allata sinefungin is a competitive inhibitor of the synthesis of methylfarnesoate with Ki of 1 μM.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 2002. / Title from PDF t.p. (viewed on Dec. 10, 2005). Vita. Includes bibliographical references (p. 70-78).

Processing information in the nervous system is energetically expensive, constraining the ability of the system to survive disturbances caused by stress. While some organisms compensate for extreme changes in the abiotic features of their environment, the mechanisms underlying this are poorly understood. We used the locust Descending Contralateral Movement Detector (DCMD) neuron to study how the propagation characteristics of action potentials (APs) change following an acute energy stress in control and heat shock (HS) pre-treated animals. We also attempted to determine if Ca2+ is involved in the DCMD AP and the possible changes indicated above. Conduction velocity decreased over an hour of recording in all groups, except those with minimal dissections, and we observed an increase in AP half-width and a decrease in the slope of the rising phase of the AP over time. After HS pre-treatment the response to a standard looming stimulus was delayed, showed significantly fewer APs and a lower peak frequency compared to controls. Brief application of sodium azide (NaN3) as an acute metabolic inhibitor did not subsequently affect DCMD’s conduction velocity or ability to fire at high frequencies during the recording period. There were no significant differences from control animals with extracellular Ca2+ manipulations; however we cannot conclude that Ca2+ does not contribute to DCMD’s AP because Na+ could have flowed through Ca2+ channels in the absence of extracellular Ca2+. Furthermore, examination of possible performance impairments with decreased Ca2+ currents, to indicate if Ca2+ current manipulation may account for the performance impairment, could not be conducted because no differences in AP characteristics were observed with Ca2+ manipulations. We suggest that the slowing of propagation in all groups represents a response to energetic stress and that HS modifies neuronal properties in ways that can be interpreted as saving energy in case of future stressors. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2009-08-11 08:36:26.571

Spreading depression (SD) is an interesting and important phenomenon due to its role in mammalian pathologies such as migraine, seizures, and stroke. Until recently investigations of the mechanisms involved in SD have mostly utilized mammalian cortical tissue, however in my thesis I demonstrated that SD-like events occur in the CNS of an invertebrate model, Locusta migratoria. Locusts enter comas in response to stress during which neural and muscular systems shut down until the stress is removed, and this is believed to be an adaptive strategy to survive extreme environmental conditions. Using the ventilatory central pattern generator (vCPG) as a model circuit I was able to show that stress-induced arrest of vCPG function is associated with SD-like events in the locust metathoracic ganglion (MTG) that closely resemble cortical SD (CSD) in many respects, including mechanism of induction, extracellular potassium ion ([K+]o) changes, and propagation in areas equivalent to mammalian grey matter. SD-like events in the locust were characterized as abrupt [K+]o increases associated with electrical activity silence in the locust CNS that propagate to other areas within the MTG. In this thesis I described the generation of comas by several cellular stressors (hyperthermia, metabolic stressors, Na+/K+-ATPase inhibition, and KCl) and the associated SD-like events in the locust, provide a description of the similarities to CSD, and show how they can be manipulated both by stress preconditioning and pharmacologically. I showed that hyperthermic vCPG arrest can be preconditioned by prior heat shock (HS) treatment and induced-thermotolerance was associated with an increased rate of [K+]o clearance associated with vCPG recovery that was not linked to changes in ATP levels or total Na+/K+-ATPase activity. I also provided evidence for the involvement of the stress-sensor AMP-activated protein kinase (AMPK) in stress-induced comas in the locust. AMPK activation was linked to a switch in motor pattern behavior following recovery from anoxia-induced vCPG arrest and exacerbated repetitive SD-like events induced by ouabain (Na+/K+-ATPase inhibitor). I suggested that locust SD-like events are adaptive by conserving energy and preventing cellular damage, and I provided a model for the mechanism of SD onset and recovery in the locust nervous system. / Thesis (Ph.D, Biology) -- Queen's University, 2010-08-05 16:08:19.905

No description available.

Previous studies on the regulation of salt and water reabsorption in the insect excretory system have concentrated on the rectum, while regulation of the ileum has received little attention. Cl⁻ transport is the predominant ion transport process in both the ileum and rectum of the desert locust and drives fluid absorption. The central nervous system (CNS) was surveyed for factors which stimulate Cl⁻-dependent short-circuit current (I[formula omitted]) using in vitro flat sheet preparations of locust ileum as a bioassay. All ganglia extracts tested (except the corpora allata) caused significant increases in ileal I[formula omitted]. Extracts of muscle tissue, used as a control, had no effect on ileal I[formula omitted] indicating that stimulants were not general metabolites present in locust tissue. Crude extracts of the corpus cardiacum (CC) and fifth ventral ganglion (VG5) stimulated ileal I[formula omitted] in a dose-dependent manner and both caused an increase in K⁺ and Na⁺ absorption as previously observed with cAMP. CC and VG5 had no effect on ileal NH₄⁺ secretion but both inhibited ileal H⁺ secretion. Most of the stimulatory effects of CC and VG were largely abolished by treatment with trypsin and chymotrypsin, suggesting that the stimulants were peptides. CC and VG5 factors were apparently separate compounds because they differed in the time courses of ileal I[formula omitted] response, thermal stability, and extraction properties. Reversed-phase high performance liquid chromatography (RP-HPLC) of water extracts of CC identified two distinct factors (fractions D and F) which stimulated ileal I[formula omitted] and a third factor (fraction G) which had little effect on I[formula omitted], but which caused a five-fold increase in ileal fluid transport (J[formula omitted]). None of these fractions increased rectal J[formula omitted]; moreover, fraction D stimulated rectal I[formula omitted] at higher doses. These results provided the first indication that separate stimulants act on locust rectum and ileum. The most potent factor in CC acting on ileal I[formula omitted] was isolated using a four-step purification procedure, utilizing C₈ and phenyl RP-columns for separation. Amino acid analysis of this purified peptide indicated a molecular weight of 7700 daltons and a near complete amino acid sequence (50 out of 65) was determined. The purified factor (S. gregaria ion transport peptide; ScgITP) was assayed on all ileal ion transport processes influenced by crude CC extracts. ScgITP caused quantitatively the same range of effects as crude CC extracts, in that it stimulated Cl⁻, K⁺, and Na⁺ reabsorption and inhibited H⁺ secretion. High doses of ScgITP (5 CC equiv.ml⁻¹) caused the same maximum response on all these systems as crude CC extracts (0.25 CC equiv.ml⁻¹). ScglTP is unlikely to be chloride transport stimulating hormone, previously reported to act on the rectum, because a maximum rectal I[formula omitted] response was not achieved and there was no effect on rectal J[formula omitted], which is Cl⁻-dependent. It appears that ScgITP acts through cAMP as the second messenger to stimulate reabsorptive processes because this cyclic nucleotide mimicked the actions of ScgITP and crude CC extracts. In support of this view, ileal I[formula omitted] was also stimulated to maximum levels by 5mM theophylline and 50μM forskolin. The inhibition of H⁺ secretion by ScgITP must occur through a different intracellular pathway because this action was not mimicked by cAMP. / Science, Faculty of / Zoology, Department of / Graduate

The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. In the absence of exogenous CO₂, HCO₃⁻, and phosphate, the isolated rectum (under both open- and short-circuit current conditions) was capable of rates of net acid secretion (J[subscript]H+) similar to those observed in vivo, demonstrating the viability of the preparation and suggesting that rectal acidification was due to proton secretion rather than selective movements of HCO₃⁻ or phosphate. The possibility that trace levels of metabolic CO₂ might be generating sufficient HCO₃⁻ to account for the observed rates of rectal acidification (via HCO₃⁻ reabsorption) was assessed by adding exogenous CO₂/HCO₃⁻ to the contraluminal bath. The small increases in J[subscript]H+ observed after addition of 2% or 5% CO₂ were shown to be due to simple hydration of CO₂ which had diffused into the lumen (from the contraluminal bath), rather than changes in rates of HCO₃⁻ reabsorption. Since measurable quantities of luminal HCO₃⁻ did not directly affect the apical acid/base transport mechanism per se, it was concluded that metabolic CO₂ could not generate sufficient HCO₃⁻ in the lumen to account for the rates of rectal acidification observed under nominally CO₂/HCO₃⁻-free conditions and that J[subscript]H+ must be due to a proton secretory rather than bicarbonate reabsorptive mechanism. Microelectrode measurements of intracellular pH (pHi) and apical and basolateral membrane potentials (Va and Vb respectively) indicated that luminal pH was not in equilibrium with either contraluminal pH or pHi and that the mechanism responsible for active luminal acid secretion resided on the apical membrane. Preliminary measurements of bath total ammonia (ie. NH₃ + NH₄+) levels in the previous experiments suggested that the rectum was actively secreting ammonia at significant rates across the apical membrane into the lumen. If the ammonia crossed the apical membrane as NH₃ rather than NH₄+, rates of luminal ammonia secretion (J[subscript]Amm) would have to be added to J[subscript]H+ to obtain corrected values of luminal proton secretion. In the absence of exogenously added ammonia and CO₂, ammonia was preferentially secreted into the lumen under both open- and short-circuit current conditions. J[subscript]Amm was dependent on the presence of luminal amino acids and was relatively unaffected by K[superscript]+ removal or changes in luminal pH from 7.00 to 5.00. Bilateral Na+ substitution or luminal addition of ImM amiloride reduced J[subscript]Amm by 63% and 65% respectively. The data consistently demonstrate that the rectum secretes significant quantities of endogenously produced ammonia preferentially into the lumen as NH₄+ rather than NH₃ via an apical Na[superscript]+/NH₄[superscript]+ exchange mechanism. Clearly, rates of net acid secretion estimated by titratable acidity do not have to include a correction for luminal ammonia secretion. Although J[subscript]H+ was completely unaffected by changes in contraluminal pH, it could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J[subscript]H+ was not dependent on Na[superscript]+, K[superscript]+, CI⁻, Mg₂+, or Ca+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of J[subscript]H+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na[superscript]+/H[superscript]+ exchange mechanism. Inhibition of JH+ by anoxia or reduction of luminal pH unmasked a significant proton diffusional pathway on the apical membrane in parallel with the active proton pump. The fact that J[subscript]H+ was significantly inhibited (42%-66%) by contraluminal addition of ImM cAMP and relatively unaffected by changes in contraluminal pCO₂ or pH suggests that net acid secretion in the locust rectum in vivo is modulated by circulating hormonal factors rather than haemolymph pH or pCO₂ per se. / Science, Faculty of / Zoology, Department of / Graduate

This thesis addresses two hypotheses: 1) ooplasmic ion activity is regulated during water uptake by locust eggs, and, 2) activities of Ca⁺⁺ and H⁺ are maintained at levels appropriate for their use as signals for developmental processes, including activation, in insect eggs. Hypothesis 1 is based on the perturbing effect of large changes in intracellular Na and/or K activity on enzyme function in eukaryotic cells. Hypothesis 2 is based on the ionic hypothesis of activation developed from studies of eggs of marine invertebrates. Electrical potential difference across egg membranes (PD_egg), and ooplasmic activities of Na⁺, K⁺, Cl⁻, Ca⁺⁺ and H⁺ were measured with double-barrelled ion-selective microelectrodes. Locust eggs maintained a measurable potential difference across egg membranes throughout development. Input resistance (Rᵢₙ) decreased by approximately 5-fold in eggs after fertilization suggesting that the chorion is not the major barrier to ion movements into and out from the egg. Chilling and anoxia decreased the contribution of a metabolic component to PD_egg. The effects of hypercapnia on PD_egg and ooplasmic pH suggest that the metabolic component may be a H⁺-pump. Chloride diffusion contributes to PD_egg in eggs after fertilization; contributions of other ions were relatively small. Potential differences measured in internally perfused locust eggs after osmotic lysis of the serosal epithelium indicated that the chloride-dependent component of PD_egg is developed across the chorion and/or serosal cuticle. In vivo values of PD_egg are probably less negative than values measured in control saline because chloride content of ground water is low. Ooplasmic sodium and potassium activities remained at typically intracellular levels during water uptake, possibly due to release from internal stores. Chloride activities were typical of extracellular fluids and were not regulated during water uptake. Measurements of pH and pea in locust eggs are consistent with the increases in these parameters predicted by the ionic hypothesis of activation. calcium activity in the ooplasm of unfertilized locust eggs (pCa 6.4 -4.9) appeared to be at a level appropriate for the use of calcium as a signal or second messenger. Calcium activity increased 100fold within 1 day of fertilization, and 1000-fold by day 3. Calcium entry from external sources at fertilization and release from internal stores later in development may contribute to the progressive increase in ooplasmic Ca⁺⁺ activity. The ooplasm likely alkalinizes after oviposition as ambient pCo₂ declines. Available data suggest a metabolically-dependent proton pump may control ooplasmic pH in locust eggs, in contrast to the Na⁺/H⁺ exchanger implicated in alkalinization of marine invertebrate eggs. / Thesis / Master of Science (MS)