Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

Proteolytic enzymes and blood digestion in the mosquito,Culex nigripalpus

The synthesis of proteolytic enzymes in the fat body and midgut of female Culex nigripalpus was followed. The effects of brain factor(s) and RNA levels in the fat body were correlated with the synthesis of proteolytic enzymes. Trypsinlike activity in the midgut of C. nigripalpus accounted for 80% of total proteolytic activity, whereas chymotrypsinlike activity accounted for 5–7% of total proteolytic activity. Synthesis of porteases in the midgut and fat body reached a peak at 35 h and 22 h after the blood meal, respectively. In the fat body, proteolytic enzyme activity fell to a low level 30 h after the blood meal, but activity in the midgut reached a low level 58 h after the blood meal. The presence of low protease activity in the fat body at the time of peak vitellogenin synthesis indicated that processing of vitellogenin was not done in this tissue. Fat bodies incubated in vitro in the presence of [¹⁴C]valine synthesized a [¹⁴C]labeled trypsinlike molecule identified as such with antitrypsin antibodies and specific substrate p-toluene-sulphonyl-L-arginine methylester (TAME) and on disc gel electrophoresis in the presence of dodecyl sulfate. The sizes of the proteins found inside and outside the peritrophic membrane were determined by gel-chromatography and disc gel electrophoresis in the presence of dodecyl sulfate. The molecular weight (± SEM) of the largest polypeptide that migrated through the peritrophic membrane into the ectoperitrophic space was found to be 23,000 ± 2,000 daltons. Based on these results, a model is proposed to account for blood digestion in the mosquito midgut, along with the role of the peritrophic membrane.     

Lipase activity and stimulation mechanism of esterases in the midgut of female Aedes aegypti

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.     

Effects of blood and virus-infected blood on protein expression in the midgut of the dengue vector Aedes albopictus

Although the mosquito midgut is the primary site of bloodmeal storage and the first line of defence against pathogenic infection, little is known about its proteic composition at a time when an increasing number of proteins are reported to impair viral infection. Aedes albopictus Skuse (Diptera: Culicidae) is an important vector of the dengue virus. We compared 2-dimensional protein profiles of the adult midgut in this species, taking into account bloodmeal status. The comparison of profiles from sugar-fed and blood-fed females showed that a considerable number of proteins were present in both midguts. In addition, one set of proteins was present only after sugar intake and another set only after blood intake. The comparison of profiles of blood-fed midguts and dengue virus-2 infected blood-fed midguts revealed that at least six proteins were present only in the infected midguts. These results are discussed in the context of the identification of midgut proteins involved in the dengue virus infection process.     

Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo : rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker

Background

Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite.

Methods

We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood.

Results

Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females.

Conclusion

Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.     

Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes

Injection of partially purified oostatic hormone (0.7 μg) into female Aedes aegypti inhibited egg development, proteolytic enzyme activity, and blood digestion in the midgut, whereas control injections of saline or insulin chain A (0.7 μg) did not affect these processes. Oostatic hormone given by enema, on the other hand, did not inhibit proteolytic enzyme activity, indicating that the hormone acts outside the midgut. A single injection of oostatic hormone (0.7 μg) caused a 1.7–1.5-fold reduction in activity of trypsinlike enzymes during blood digestion, with a 10-h delay in peak activity. Using [1,3-³H]diisopropylfluorophosphate (DFP) in the presence of 8 mM tosylamide-2-phenylethyl chloromethyl ketone, the synthesis of trypsinlike derivatives was followed in the midgut of female A. aegypti. A 4-fold reduction in [1,3-³H]diisopropylphosphoryl-trypsinlike derivatives was noted after oostatic hormone treatment. Several isozymes that are normally synthesized were absent in the presence of DFP, as assessed by polyacrylamide gel electrophoresis. Injection of oostatic hormone into decapitated and ovariectomized females that did not synthesize ecdysteroids inhibited trypsinlike enzyme synthesis and blood digestion in the midgut, indicating that oostatic hormone inhibits the midgut cells and not the ovary or the brain's endocrine system. Comparison between oostatic hormone and soybean trypsin inhibitor indicated that the former inhibited trypsin synthesis whereas the latter inhibited trypsin activity. A. aegypti oostatic hormone is not species specific and injections of the hormone into Culex quinquefasciatus, Culex nigripalpus, and Anopheles albimanus caused inhibition of egg development, blood digestion, and synthesis of trypsinlike enzymes. A direct relation between oostatic hormone synthesis and the regulation of trypsinlike activity in the midgut is proposed.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

PHARMACOLOGICAL REGULATION OF DIGESTION IN THE ANAUTOGENOUS FLESH FLY,Sarcophaga crassipalpis,BY SIMPLE INJECTION OF 6‐HYDROXYDOPAMINE

Female anautogenous Sarcophaga flesh flies need a protein meal to start large‐scale yolk polypeptides (YPs) production and oocyte maturation. Protein meal rapidly elicits a brain‐dependent increase in midgut proteolytic activity. Trypsin and chymotrypsin together represent over 80% of protease activity in liver‐fed flies. Abdominal injection of 6‐hydroxydopamine (6‐OHDA) dose‐dependently prohibits this increase in proteolytic activity at translational level in a similar way as post liver feeding decapitation. Delayed injection of 6‐OHDA later than 6 h post liver meal has no effect. In flesh flies, chemical decapitation by 6‐OHDA, by interrupting the brain‐gut dopaminergic signaling, can be used as tool for the controlled inhibition of midgut proteolytic activity and subsequent ovarial development. Inhibition of ovarial development is probably indirect due to a deficit in circulating amino acids needed for YPs synthesis.     

An hypothesis of the mechanism controlling proteolytic digestive enzyme production levels in Stomoxys calcitrans

Flies fed a human blood meal and sacrificed 9 h later were assayed to give information on unfed fly weight, meal weight, total midgut protein, total midgut proteolytic activity, anterior midgut protein, anterior midgut proteolytic activity, posterior midgut protein, and posterior midgut proteolytic activity; correlation coefficients were calculated for all pairings of these parameters. Posterior midgut protein showed a positive correlation with posterior midgut proteolytic activity and on this evidence it is concluded that proteolytic digestive enzyme secretion in the midgut of Stomoxys calcitrans is controlled by a secretogogue mechanism.It is proposed that the only direct stimulus the food supplies in the control of digestive enzyme production is that for digestive enzyme release from the production cells. It is also proposed that the basis of the secretogogue mechanism is that digestive enzymes are produced in direct proportion to the quantities of amino-acids available for their synthesis and that this is a consequence of the quantities of amino acids released from the food during digestion.     

Proteolytic activity in the ectoperitrophic fluid of blood-fed Culex nigripalpus

In blood-fed Culex nigripalpus Theobald, proteolytic activity appeared in the ectoperitrophic fluid after 3 h, but only after 6 h in a homogenate of the blood-filled midgut. The activity continued to be higher in ectoperitrophic fluid than in whole gut homogenate until about 40 h after the meal, when most of the intact clot had disappeared. Apparently, undigested blood inhibits proteolytic activity. The blood clot lacked activity and the inhibitor could not be removed by washing. The results are compatible with a hypothesis that the peritrophic membrane separates the digestion from the ingestion compartment.     

Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.     

Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme(s) in the midgut of Glossina morsitans

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.     

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.     

Digestive processes of haematophagous insects: control of trypsin secretion in Glossina morsitans

Glossina morsitans females were fed upon goats or components of beef blood through an Agar/Parafilm membrane and for each fly the following were determined: fly weight, meal weight, posterior midgut trypsin, posterior midgut protein, anterior midgut trypsin, and anterior midgut protein. Secretion of trypsin was stimulated by feeding flies upon goats, defibrinated beef blood, beef serum, haemolysed beef erythrocytes but not washed beef erythrocytes. There was a significant correlation between posterior midgut trypsin and the amount of protein in the posterior midgut, and the slope of the regression of trypsin upon protein content was significantly different from zero. There was a significant correlation between posterior midgut trypsin and meal size for flies 0 to 24 hr after emergence, but not those 24 to 48 hr old when fed upon a goat. For unfed flies there was a significant correlation between posterior midgut trypsin and fly weight.     

Chymotrypsin inhibitors in mosquitoes: Activity profile during development and after blood feeding

Chymotrypsin and trypsin inhibitors persist throughout all developmental instars of Aedes aegypti. After a blood meal, inhibitor activity against chymotrypsin was more than double that of sugar-fed females, but only weak activity was detected in midguts where proteinase inhibitors has been thought to regulate proteinases during blood digestion. A fourfold increase in the ratio of abdominal/thoracic inhibitor activity after the blood meal strongly suggested that fat body, or other abdominal tissues, represent the major source of inhibitor. Chymotrypsin inhibitor activity was deposited in maturing oocytes. Similar results were obtained with blood-fed Anopheles albimanus. Chymotrypsin inhibitor was active against different mosquito proteinases and against bovine α-chymotrypsin and trypsin, but not against subtilisin, pancreatic elastase, or fungal proteases; chymotrypsin inhibitors did not interfere with bacterial growth. The hypothesis on the regulation of blood digestion through the action of proteinase inhibitors during the gonotrophic cycle was abandoned and its involvement in the phenoloxidase cascade in the mosquito egg chorion is suggested instead. Arch. Insect Biochem. Physiol. 36:315–333, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.