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The distribution of acetylcholine and unspecific esterases in the midgut of female Aedes aegypti L
Abstract
1.1. The esterases of the midgut epithelium and their secretion into the gut lumen were studied in mosquitoes.2.2. Significant differences in the esterase patterns of the midgut epithelium of newly emerged, sugar-fed and blood-fed females were found.3.3. A high increase of the total esterase activity and of acetylcholinesterases can be observed in the midgut epithelium after a blood meal.4.4. Secretion of esterases takes place after blood intake including most of the enzymes occurring in the epithelium. High amounts of acetylcholinesterases are found.5.5. The results are discussed in connection with the known facts of lipid metabolism.
... Recently, Hernandez and Wheelock (2009) reported up to twelve CbE isozymes in the gastrointestinal tract, body wall muscle and reproductive tissues of L. terrestris, being particularly abundant in the crop, gizzard and foregut. The abundance of CbEs in the gastrointestinal Comparative Biochemistry and Physiology, Part C 150 (2009) 503–511 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c tract of earthworms leads to hypothesize if these enzymes represent an enzymatic barrier for reducing pesticide uptake from contaminated ingested soil, or even to speculate if these esterases can be secreted into the gut lumen by earthworms themselves as this occurs with other invertebrates such as spiders (Mommsen, 1978), lepidopterans (Turunen and Chippendale, 1977), mosquitoes (Geering and Freyvogel, 1974) or cockroaches (Cook et al., 1969). The aims of this study were therefore to determine the presence of CbEs in the earthworm gut content and to investigate whether gut wall is a source of luminal CbE activity. ...
... For example, Turunen and Chippendale (1977) found several CbE isozymes in the midgut fluids of the larval southwester corn borer (D. grandiosella), which were sensitive to 10 − 5 M eserine and 10 − 4 M dichlorvos. Similarly, Geering and Freyvogel (1974) showed an unspecific esterase activity that increased its hydrolytic activity in the midgut lumen of female mosquitoes (Aedes aegypti) after blood ingestion; although such an increase was not due to blood esterases from the host. They suggested that secretion of these esterases could be involved in the lipid metabolism and mobilization. ...
Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.
... The digestion ability is mainly characterised by lysosome and rough endoplasmic reticulum. The metabolic activity of these organelles increases after a blood-meal due to the synthesis of digestive enzymes [Rossignol et al., 1982] like esterase [Geering and Freyvogel, 1974] and α-glucosidase [Billingsley and Hecker, 1991] which also are present in the lumen of the MG. Proteolytic enzymes such as trypsin, aminopeptidase and so on are mostly secreted to digest the bloodmeal. ...
In eco‐evolutionary studies of parasite‐host interactions, virulence is defined as a reduction in host fitness as a result of infection relative to an uninfected host. Pathogen virulence may either promote parasite transmission, when correlated with higher parasite replication rate or decrease the transmission rate if the pathogen quickly kills the host. This evolutionary mechanism, referred to as ‘trade‐off’ theory proposes that pathogen virulence evolves towards a level that most benefits the transmission. It has been generally predicted that pathogens evolve towards low virulence in their insect vectors, mainly due to the high dependence of parasite transmission on their vector‐survival. Therefore the degree of virulence which malaria parasites impose on mosquito vectors may depend on several external and internal factors. Here we review briefly (I) the role of mosquito in parasite development, with a particular focus on mosquito midgut as the battleground between Plasmodium and the mosquito host. We aim to point out (II) the histology of the mosquito midgut epithelium and its role in host defence against parasite's countermeasures in the three main battle sites, namely a‐ the lumen (microbiota and biochemical environment) b‐ the peritrophic membrane (physical barrier), and c‐ the tubular epithelium including the basal membrane (physical and biochemical barrier). Lastly, (III) we describe the impact which malaria parasite and its virulence factors have on mosquito fitness. This article is protected by copyright. All rights reserved
... Nevertheless, some authors postulate that feeding activity, hormone-related mechanism, or the presence of lipid-rich materials in diet are among the potential factors contributing to blood esterases interindividual variations (Rattner and Fairbrother 1991; Thompson 1993). Past studies show that CbE activity plays a notable role in the metabolism of lipids in invertebrates (Geering and Freyvogel 1974; Mommsen 1978). Similar findings have been documented in rats (Wassmer et al. 1988) and mice (Van Lith et al. 1991, 1992) fed with lipid-rich diets. ...
In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring.
Forty-two T. merianae individuals were grouped by sex and size to identify potential differences in their enzyme levels to allow for proper representation of normal values for females, males, juveniles, and hatchlings. Mean CbE was determined using two model substrates: alpha-naphtylacetate (α-NA) and p-nitrophenyl valerate (4-NPV). BChE and CbE sensitivity to malaoxon (Mx) was also evaluated as well as the possibility of BChE reactivation with pyridine-2-aldoxime methochloride (2-PAM).
Mean adult females' BChE was significantly higher than adult males, juveniles, and hatchlings. No significant differences were found between groups regarding CbE. CbE (4-NPV) activity showed slightly negative correlation with lizard snout-vent length, while BChE and CbE (α-NA) showed no correlation with body size. Apparent IC(50) values for BChE and CbE (α-NA) suggested different sensitivities among groups. CbE (4-NPV) could not be inhibited. All Mx-inhibited groups treated with 2-PAM in a final concentration of 2.8 mM showed clear signs of reactivation.
In conclusion, the results demonstrate that (1) plasma esterase activity did not vary with age and sex, except for BChE activity, and (2) because biological and environmental variables could be confounding factors in the response of plasma cholinesterases, complementary biomarkers like CbE inhibition and oxime-induced reactivation of esterases are strongly recommended.
... Factors such as temperature, nutritional status, reproductive stage or burrowing activity may affect the esterase activity of earthworms (Fordsmand and Weeks, 2000; Lowe and Butt, 2007 ). Particularly , nutritional status could change the CE activity because this esterase seems to metabolize lipids in invertebrates (Geering and Freyvogel, 1974; Mommsen 1978) and vertebrates (Van Lith et al., 1992), and its hydrolytic activity could be altered by ingestion of lipid materials or inanition. In this work, earthworms were not fed throughout the experiment and a nutritional deficit could explain the high variation of esterase activities; particularly gut CE. ...
Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil.
Lactate dehydrogenase (LDH) isoenzymes from Aedes aegypti were separated by acrylamide electrophoresis. Five isoenzymes were found in the head, abdomen, and thorax with those moving fastest (LDH 1 and LDH 2) located predominantly in the head. The slow moving isoenzymes (LDH 4 and LDH 5) were found in high concentrations in both the abdomen and thorax. In whole mosquito extracts, the relative activities of LDH 1 and LDH 2 decreased while the slow moving LDH 4 and LDH 5 increased in relative activity as the mosquito became older. Following a blood meal, both the relative and specific activities of LDH 1 and LDH 2 rose sharply while the specific activity of LDH 4 and LDH 5 remained constant. It seems likely that the blood meal stimulates the genetic loci which regulate the synthesis of the H subunits of LDH. However, the loci which regulate synthesis of the M subunits do not appear to be affected.
Polyacrylamide gel disc electrophoretic technique was used to examine the esterase pattern of nead, haemolymph, alimentary
canal., ovary and testis of the Uzi fly. The zymograms revealed the varied pattern of esterases both in number and type. This
varied pattern suggested several roles for these enzymes present in different tissues
Stomach cells of female Asplanchna sieboldi are specialized for absorption and intracellular digestion of nutrients. Evidence is presented to show that electron-opaque colloidal substances, present in the medium and within digestive vacuoles of the prey (Paramecium), are taken up by the stomach cells at the apical cell membrane and sequestered within food vacuoles which contain hydrolases working in both the acid and alkaline pH range. The stomach cells are also implicated in the absorption of molecules below the resolving power of the electron microscope. In rotifers possessing a complete digestive tract, this task is presumed to be handled by the intestine.
The ultrastructure of the midgut of Aedes aegypti females was investigated by means of morphometric techniques. The interest was focussed on quantitative differences in the composition of epithelial cells between the anterior (A) and posterior (P) part of the midgut. The aim was to attribute defined functions to each of these 2 parts.
5 different physiological stages were compared: 1 = immature female; 2 = ready for first blood meal (b.m.); 3 = digestion of b.m.; 4 = ready for 2nd b.m.; 5 = aging female.
Whereas in stage 1, when the gut has not yet reached a functional state the epithelial cells of A and P resemble each other, distinct differences develop in subsequent stages. Generally the cell volume and the nuclear-cytoplasmic ratio become higher in P than in A. Concomitantly the mitochondrial content and the surface area of the rough endoplasmic reticulum are more prominent in P than in A. On the other hand more microvilli, a more extensive basal labyrinth and a well developed smooth endoplasmic reticulum are present in A.
Enlarged cytoplasmic volumes, especially high in P, are found 24 h after the first b.m. (stage 3). In accordance with this increase the volume of organelles and/or their membrane surface areas also reach maximal values. This augmentation of the metabolic cell apparatus suggests that during blood digestion the whole organ develops an increased functional capacity.
From the quantitative structural differences in A and P we concluded that the A-part of the midgut is especially involved in absorption. To the P-part more protein synthesis may be attributed, thus indicating an enhanced formation of digestive enzymes.
Although this study deals with a rather specialised insect (blood digestion), it appears that our hypothesis attributing functions to defined midgut zones, agrees to a certain extent with the “secretion-absorption-model” of Berridge (1970).
The effects of various parameters on the nonspecific esterase electropherograms of the Knights Landing strain of the mosquito Culex tarsalis were determined by isolelectric focusing electrophoresis in acrylamide gels. The parameters examined were substrate preference, isolation/incubation media composition, tissue distribution, aging, deoxycholate stimulation and acetylthiocholine hydrolysis. The results corroborate the identity of two major acetylcholinesterases and document a single lipase in this mosquito. The nonspecific esterases of C. tarsalis can now be classified as: one acetylesterase, one arylesterase, one lipase, two acetylcholinesterases and thirteen carboxylesterases.
The activities of esterases, hydrolyzing (Acnp), of several strains of the mosquito, Culex tarsalis, are examined spectrophotometrically. Michaelis-Menten constants were determined to be 49.5 μmole/10 min () and 1.4 mM (). Mg2+ ions stimulate the reaction much above other divalent cations (Ba, Ca, Co, Cu, Mn and Fe) with a maximal activity at 33 mM. The optimal temperature is 30°C, with a range of approximately 1°C depending on the mosquito strain. The efficacy of adding deoxycholate to the isolation medium is examined and compared to H2O and buffer. Inhibition data and isoelectric focusing electrophoresis indicate that, with Acnp as the substrate, 50–55% of the measured activity is attributable to an arylesterase.
Chemical control of pests is still necessary in agriculture, despite the growing efforts to introduce biocontrol-based strategies. Many studies have evidenced the harm of pesticide side-effects on natural populations of pest enemies and other non-target organisms. Moreover, pesticide-contaminated soils can act as a secondary pollution source causing contamination in environmental compartments of critical concern to public health (water resource). These environmental risks need to be assessed and monitored for decision-making related to the post-authorization management of pesticides. Under these considerations, earthworm esterases can be a suitable tool for these regulatory and environmental purposes. Herein, it is suggested the use of earthworm esterases as biomarkers to be included in a field toxicity test. Furthermore, the potential role of gut carboxylesterases (CEs) in the modulation of pesticide toxicity is discussed in view of their contribution to the natural tolerance of earthworms to pesticides, and consequently the appropriate selection of earthworm species for regulatory toxicity testing. Finally, it is postulated that CE secretion into the earthworm gut could be an environmental friendly methodology in the enzymatic bioremediation of pesticide-contaminated soils. (C) Pesticide Science Society of Japan
1. The nonspecific esterases of the mosquito, Culex tarsalis, were examined through conventional and isoelectric focusing acrylamide gel electrophoresis. 2. Conventional acrylamide gel electrophoresis resolved five components. These were characterized as: three carboxylesterases, one acetylcholinesterase and one acetylesterase. 3. Isoelectric focusing resolved 18 components. These were characterized as: 14 carboxylesterases, two acetylcholinesterases, one acetylesterase and one arylesterase. 4. The reproducibility and reliability of isoelectric focusing is discussed and compared to conventional acrylamide gel electrophoresis for the examination of multi-component isozyme systems such as non-specific esterases.
Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze alpha- and beta-naphthylacetate and alpha-naphthylpropionate with almost equal capacity. alpha-Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate di-isopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.
A triglyceride-splitting esterase was identified in the midgut of sugar-fed and of blood-fed mosquitoes. Maximal activity is reached 15 hr after a blood meal.Enema experiments revealed the stimulation mechanism of acetylcholine and unspecific esterases in the midgut. Simple stretching of the epithelium causes increased enzyme production that involves the same esterases as after a blood meal. The results are discussed in connexion with the known data on the stimulation of enzymes and of the peritrophic membrane in mosquitoes.
1. In the pupal stage of the cecropia silkworm, antigen 7, a protein with the solubility characteristics of an albumin, is present in female blood in approximately a thousand times higher concentration than in the blood of males. Antigen 7 is undetectable in the blood of larvae of either sex. It first appears in the blood after the larva has spun its cocoon, and is present throughout all subsequent stages of metamorphosis. Late in the pupal-adult transformation, when the eggs are produced, the concentration of antigen 7 in female blood decreases significantly.
2. An antigen which could not be distinguished from antigen 7 immunologically is present in solution in the yolk of unfertilized eggs.
3. In females which, by ovariectomy, were prevented from forming eggs, the concentration of antigen 7 in the blood increased during the usual period of egg formation rather than undergoing the normal decrease. Ovaries transferred to the hemocoel of males produced eggs but were unable to incorporate antigen 7 in the yolk unless a detectable amount of the protein was present in the blood. The ovaries of polyphemus females which had been transfused with cecropia blood incorporated cecropia antigen 7 into the eggs they produced. These lines of evidence indicate that antigen 7 is secreted into the blood by some tissue other than the ovaries, and that it is subsequently drawn from the blood and deposited in the yolk.
4. The concentration of antigen 7 in the clear, liquid fraction of the yolk is four times higher than the maximum concentration attained in the blood during metamorphosis, and twenty times higher than that of the blood at the conclusion of egg formation. The protein thus appears to be transferred from blood to yolk against a concentration gradient.
Reserve deposits of fat and glycogen are known to serve as sources of energy for flight in various insects. Among the Diptera only glycogen has been shown to serve this purpose ; the respiratory quotient of Drosophila in flight is close to 1·0 (Chadwick, 1947), and during flight glycogen disappears from the fat body but the deposits of fat are untouched (Wigglesworth, 1949). Tabanus has also been shown to use fat body glycogen during flight while apparently making no use of the fat deposits (Hocking, 1953).
The salivary glands, midgut, and hindgut of larvae of Hypoderma bovis exhibit a unspecific esterase activity and also a proteolytic one. This proteinase splits only structural protein (scleroprotein). Diisopropylfluorphosphate inhibits the enzymatic activity of the esterases and the proteinase; Soya bean trypsin-inhibitor only stops the proteinase. The larvae regurgitate parts of the content of the midgut, which digests the food extraintestinally. Differences in inhibition and electrophoretic analysis indicate differences between the enzymes of the salivary glands and the gut.
Starch-gel electrophoresis of extracts of larval gut, haemolymph, and body wall and of adult gut and flight muscle from Pieris brassicae revealed thirteen esterases. From their reactions with inhibitors all were shown to be carboxyl esterases. Nine of the enzymes occurring in the larval gut were also found to be present in the haemolymph but at a much lower concentration. Striking changes occurred in the carboxyl esterases of gut and haemolymph at different stages of the life cycle.
The importance of mosquitoes as disease vectors is now well established, but much remains to be learned about the fundamental processes of digestion in mosquitoes, especially as they relate to the transmission of these diseases. The studies reported here deal with the stimulation of proteolytic enzyme activity in the adult female yellow-fever mosquito, Aedes aegypti L., in relation to the interval of time after feeding and other factors. METHODS The Aedes aegypti mosquitoes used in these tests were obtained as needed from the colony maintained by an Ohio State University Research Foundation Project sponsored by the U. S. Public Health Service. Female mosquitoes four to six days after emergence were used, since at that age they feed most readily on blood and have, by that time, disposed of any larval food which remained in the midgut at emergence (Fisk, 1950). The insects were transferred to a lamp chimney covered at the top with gauze and resting on a cross-slitted dental rubber dam held in place by an embroidery hoop. Blood Feeding Techniques
1. A partial hydrolysis of 14C-labelled tripalmitin has been demonstrated in the crop of Periplaneta americana L.
2. No significant absorption of tripalmitin and its derivatives could be demonstrated in the crop, whether the tripalmitin was suspended in an experimental fluid or dissolved in emulsified oleic acid.
3. Absorption took place in the mid-gut and appeared to be largely confined to the caeca and the anterior part of the ventriculus.
4. The total absorption of tripalmitin showed a linear relation with crop emptying, suggesting that the rate at which the material was allowed to leave the crop, rather than the uptake in the mid-gut, was the limiting factor in absorption.
Zone electrophoretic analysis of the gastric secretion of the cockroach, Periplaneta americana, revealed the presence of six carboxylic esterases. Two of these enzymes are resistant to 10−4 M dichlorvos (DDVP) and one, E6, is localized in the epithelium of the gastric caecum and the midgut. A purification is described for esterase E6 which yields a single zone after starch-gel electrophoresis. The purified enzyme readily hydrolysed 14C-labelled tripalmitin to free fatty acid, diglyceride, and monoglyceride, and 1-naphthyl acetate to 1-naphthol and acetic acid, and 86 per cent of both the esterolytic and lipolytic activity was inhibited by 10−3 M dichlorvos. Both the calcium and manganous ions served as activators in triglyceride hydrolysis.Three different phosphatases were isolated from the gastric fluid, one having an alkaline pH optimum and the second having an acid pH optimum (5·0) and a third having a pH optimum near 7. The acid phosphatase was inhibited by 10−3 M manganous ion; the neutral phosphatase showed a marked increase in activity in the presence of the same cation.The rôle of these hydrolases in the normal function of the insect and in the metabolism and mode of action of insecticides is discussed.
S ynopsis
An account is given of isozymes of esterase and alkaline phosphatase in females of Aedes vittatus from Italy and Rhodesia and their reciprocal hybrids.
Results of tests for digestive enzyme activity from mandibular and salivary glands and from foregut, midgut, and hindgut of major workers of the imported fire ant, Solenopsis richteri Forel, indicated a wide distribution of lipase production from these organs, thus correlating with the observed “grease‐loving” feeding habits of this insect. Amylase activity was present in both glands tested, but not detected in the gut. Both invertase and protease activities were apparently restricted to the midgut, with the protease activity being especially limited.
Zusammenfassung
VERDAUUNGSFERMENTE DER IMPORTIERTEN FEUERAMEISE , SOLENOPSIS RICHTERI (HYMENOPTERA: FORMICIDAE)
An der importierten Feuerameise wurden Untersuchungen über das Vorkommen der vier Hauptklassen von Verdauungsfermenten bei Großarbeitern (Soldaten) ausgeführt.
Die untersuchten Fermente waren Invertase, Diastase, Lipase und Protease, die untersuchten Verdauungsorgane Speichel‐ und Mandibulardrüsen, Kropf, Mitteldarm und Enddarm. Um die Ferment‐Aktivität der verschiedenen Organe zu prüfen, wurden als Substrate Sucrose, Stärke, Castor‐Öl‐Emulsion und Blutfibrin benutzt und Dünnschichtchromatographie angewandt.
Die Resultate zeigen besonders die weite Verbreitung der Lipase‐Aktivität, die der beobachteten Vorliebe der importierten Feuerameise für fettreiche Nahrung entspricht. Ebenso steht die gefundene mäßige Invertase‐ und Diastase‐Aktivität in Beziehung zu den omnivoren Fraßgewohnheiten dieser prädatorischen Insekten. Andererseits erwies sich die Protease‐Aktivität als besonders schwach; sie schien hauptsächlich auf den Mitteldarm beschränkt zu sein.
Der geringe Grad der Protease‐Aktivität in den Drüsen und im Kropf läßt vermuten, daß bei Trophallaxis den Larven das Protein‐Futter von den Arbeiterinnen in unverdautem Zustand verabreicht wird.
1.1. Five esterase isozymes were found from soluble extracts of Anopheles punctipennis.2.2. Each of the isozymes exhibited a differential specificity depending on the type of inhibitor used.3.3. The A and B loci were characterized as specific cholinesterases based on the response to treatment with a variety of organophosphates and carbamates.4.4. The other loci did not respond as uniformly to the various pesticides used in this study, but were characterized as either carboxylesterases or aromatic esterases for discussion purposes.5.5. The importance of having pure isozyme preparations is considered in conjunction with additional knowledge of the kinetic and stereospecific properties of the inhibitors.6.6. The techniques described in this work showed that mosquito isozymes may be clearly separated with a high degree of resolution.
Esterase zymograms were prepared from 14 species and strains of Culicide mosquitoes by use of starch and polyacrylamide electrophoresis. The zymograms proved to be species- and strain-specific. Variations in the zymograms of individual mosquitos were demonstrated by use of the microgel technique. This technique is described. Sexual dimorphism was shown to occur in the zymograms of one species, Anopheles stephensi. The isozymes were characterized by their reactivity toward different substrates and inhibitors. Their distribution in the mosquito's organism was studied with the whole mount technique and with organ zymograms. The stomach is particularly rich in esterase isozymes. There is a close correlation of these esterases and the lipids known to occur in the stomach epithelium and the esterases may play a role in the digestion of the blood meal. Observations on the effect of storage of samples, at low temperature and while dried on the activity of some isozymes, are reported. It is also shown that certain isozymes apparently are activated by inhibitors.
Enzyme comparisons of 12 species of mosquitoes (Diptera: Culicidae) were undertaken using the technique of starch gel electrophoresis. The 4th-stage larvae of 10 species of Aedes, 1 species of Culex, and 1 species of Anopheles were analyzed for acetate and butyrate esterases, alkaline and acid phosphatase, malic and lactic dehydrogenase.
Species-specific patterns for all 12 species were obtained with both esterase substrates and for alkaline phosphatase. Aedes aegypti (L.), and A. mascarensis (McGregor) share the same pattern for acid phosphatase and malic dehydrogenase. Both species belong in Group A, subgenus Stegomyia. A. albopictus (Skuse), A. pseudoscutellaris (Theobald), and A. scutellaris (Walker) have identical lactic dehydrogenase patterns; all occur in Group C, Stegomyia.
The overall patterns, i.e., obtained with all 6 substrates, were species specific but no clear generic differences were evident. This fact was probably a result of the small sampling of Culex and Anopheles.
Inter-specific relationships were examined by means of paired affinity indices and polygonal graphs. These instruments provided the basis for the following taxonomic inferences. A. pseudoscutellaris which is morphologically similar to A. scutellaris and supposedly more closely related to it on a phylogenetic basis, shows a higher degree of biochemical relationship to A. albopictus. On the basis of available data A. triseriatus (Say) seems to be the “biochemical type species” of the subgenus Finlaya. The placement of A. sierrensis (Ludlow) in the subgenus Finlaya on the basis of egg type was reaffirmed on a biochemical basis.
Within a few hours after injection of juvenile hormone into Hyalophora gloveri pupae, several fast-migrating carboxylesterases (EC 3.1.1.1) that are sensitive to diisopropylfluorophosphate appear in the hemolymph. Treatment of the pupae with puromycin or actinomycin D prevents the appearance of these hemolymph enzymes, suggesting de novo synthesis of the carboxylesterases. Of the several other compounds investigated, only a potent mimic of the juvenile hormone is able to induce these enzymes. When the induced enzymes are incubated in vitro with (14)C-labeled juvenile hormone, the hormone is rapidly and efficiently degraded. It is suggested that these induced carboxylesterases play an important role in the regulation of juvenile hormone titer.
1.1. Electrophoretic studies on non-specific esterases were carried out in several organs of the female mosquito, Aedes aegypti(strain Segemaganga).2.2. Esterase isozymes of the central nervous system (head), the midgut epithelium, the Malpighian tubes and the ovaries have been compared, qualitatively and quantitatively, before and mainly after a blood meal.3.3. Organ specific patterns were observed during the period of blood digestion and egg maturation, especially in the ovaries and in the Malpighian tubes. In the head the cholinesterases are increased during gonotropic cycles.
Survival data of mosquito males and females under different dietary conditions but constant antecedent and contemporaneous environmental conditions are presented. Two strains of the yellow fever mosquito Aedes aegypti (L.), a strain of the subspecies Culex pipiens fatigans Wiedemann and an autogenous strain of C. p. pipiens L. have been compared. Mean survival time and 50% survival time are considered to be more representative than maximal life span. Survival under complete starvation was significantly prolonged by feeding sucrose in all experiments, but less by feeding protein alone. In general, optimal survival was observed when males were fed on sugar and females on sugar and protein. Autogenous females of C. p. pipiens showed reduced survival upon regular blood and sugar feeding after their first gonotrophic cycle.
Recent years have seen great progress in the purification and characterization of cholinesterases. Investigation has indicated the existence of two principal groups: a fairly homogeneous group of acetylcholinesterases and a group of enzymes that utilize butyrylcholine, propionycholine, or benzoylcholine as substrates and that differ widely in their properties.This paper reviews the different types of cholinesterase and their sources, the importance of a proper choice of substrate in cholinesterase studies, methods for the purification of cholinesterases, and some of the properties of these enzymes.
Zusammenfassung Unspezifische Esterasen wurden während des Adultlebens vonAedes aegypti (L.) quantitativ bestimmt und zwischen Weibchen von 2 Stämmen verglichen. Alterungist begleitet von einer Zunahme der Bänderzahl und einer Abnahme der Esterasenaktivität pro Weibchen; die relative Aktivität der einzelnen Esterasen-Isozyme bleibt aber mehr oder weniger konstant. Nach einem Blutmahl, dem Beginn eines neuen gonotrophen Zyklus, wurden auffällige Veränderungen im Bändermuster festgestellt. 4 Tage später, nach erfolgter Eiablage, ist dieses Muster wieder ähnlich wie vor der Blutaufnahme.
Quantitative determinations of the esterase activity of the posterior midgut of the Calliphora female showed this part of the gut to possess considerable esterase activity.Calculated on a weight basis no significant difference could be demonstrated between the esterase activity of posterior-midguts with many big ‘caps’ from protein-flies and that of posterior midguts with few small ‘caps’ from sugar-flies.
The study of toxicology and other related fields has been largely based on in vitro techniques. These methods have provided quantitative information on the effects of inhibitors on enzymes, but none on the localized effects of inhibitors on selected sites of action within the animal. Histochemical study of frozen sections does provide data on the site of action of toxicants. The utility of histochemistry in conjunction with in vitro methods is discussed.The substrates acetylthiocholine and phenyl thioacetate were utilized in demonstrating cholinesterase. Neither substrate penetrated well into freshly dissected nerve cord preparations, but both compounds were hydrolysed by sectioned tissue. The leaving group of phenyl thioacetate was demonstrated to be benzenethiol. In general, acetylthiocholine was hydrolysed slightly more rapidly by insect cholinesterases. A unique cholinesterase was found in motor end-plates of cricket muscle, which hydrolyses acetylthiocholine and which was inhibited by physostigmine. No other insect muscle preparation showed this activity. Topical application of insecticides showed that a vital site of action in flies is the peripheral area of the thoracic ganglia and that in crickets the brain and nerve cord are involved at knock-down. Kinetic data indicate that acetylthiocholine has a greater affinity than does phenyl thioacetate for a variety of enzyme sources. Ultrastructural evidence shows that cholinesterases that hydrolyse acetylthiocholine are membrane-bound. Phenyl thioacetate was found to be useful as a model in designing new insecticides.
Zymograms of single individuals of Aedes aegypti were obtained by means of starch gel electrophoresis, using alpha-naphthyl acetate as substrate. Inbred lines gave consistently homogeneous patterns; earlier results from random-breeding laboratory strains had shown considerable variability. Six distinct bands were observed. The furthest moving band, designated Esterase 6, showed differential migration in two inbred lines. Reciprocal crosses between these lines gave F1 progeny showing both bands. Backcrosses of F1 to either parental line gave a 1:1 segregation. These results are consistent with the hypothesis that the two forms of Esterase 6 are controlled by a single pair of codominant alleles at a single gene locus (Est 6
a and Est 6
b). Linkage tests with marker genes have demonstrated that Est 6 is on linkage group 2, with the following alignment: spot-abdomen (9.01.0) yellow-larva (17.41.3) Est 6. Crosses with another inbred line demonstrated a third band with intermediate mobility, designated Est 6
c. An additional electrophoretic variant which seems to have a simple Mendelian basis was found in esterase band 1.
THE fat body in an insect commonly contains a single cell type which is functionally diverse. At various times it stores and mobilizes fat, protein and glycogen which it may have synthesized or sequestered. In general the fat body becomes filled with reserves as metamorphosis approaches1. Larvae of Aëdes fed on casein build up reserves of protein granules in the fat body2, and in Drosophila larvae, protein granules are induced to form in the middle of the third stadium by a hormone from the ring gland3. In Calpodes larvae the protein granules are probably translocated endocuticle. They arise suddenly within the Golgi vesicles 30-35 h before pupation, at a time when the thick larval endocuticle is being resorbed by the moulting fluid. At about 10 h before pupation other granules containing both protein and ribosomes arise from the isolation of endoplasmic reticulum in cytolysomes4. Although the formation and occurrence of protein granules in the fat body are well documented, there is little information on the origin of the protein.
Zur Histochemie und Histologie des Mitteldarms von Aedes aegypti und Anopheles stephensi im Zusammenhang mit der Blutverdauung The influence of the fungicide merthiolate on insect esterases in disc electrophoresis, ft. Histochem. Cytochem. 21, 834
- K Geering
- T A Fre 'yvogel Gander E
- K Holter
- Thomsen E
K. GEERING AND T. A. FRE'YVOGEL GANDER E. (1968) Zur Histochemie und Histologie des Mitteldarms von Aedes aegypti und Anopheles stephensi im Zusammenhang mit der Blutverdauung. Acta tropica 25, 134-175. GEERING K. (1973) The influence of the fungicide merthiolate on insect esterases in disc electrophoresis, ft. Histochem. Cytochem. 21, 834. HOLTER K. & THOMSEN E. (1971) Esterase activity of the posterior midgut of the female blowfly Calliphor a erythrocephala..7. Insect Physiol. 17, 1471-1478.
Sequestration of protein by fat body of an insect Eleetrophoresis System Digestive enzymes of the imported fire ant, Solenopsis richteri (Hymenoptera: Formicidae) Some hydrolases and their involvement in insecticide resistance
- Locke M V Collins J
- B L Ricks
- S B Vinson
LOCKE M. & COLLINS J. V. (1966) Sequestration of protein by fat body of an insect. Nature, Lond. 210, 552-553. ORTEC Instruction Manual 4200 (1970). Eleetrophoresis System. RICKS B. L. & VINSON S. B. (1972) Digestive enzymes of the imported fire ant, Solenopsis richteri (Hymenoptera: Formicidae). Ent. exp. appl. 15, 329-334. SUDDERUDDIN K. I. & TAN K. H. (1973) Some hydrolases and their involvement in insecticide resistance. Pans 19, 24-35.
Zur Histochemie und Histologie des Mitteldarms von Aedes aegypti und Anopheles stephensi im Zusammenhang mit der Blutverdauung
- Gander