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.     

Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

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.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

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.     

The synthetic pathway of trypsin in the mosquito Aedes aegypti L. (Diptera: Culicidae) and in vitro stimulation in isolated midguts

Mosquito trypsin is synthesized in vivo and in vitro in two groups of forms with differing molecular sizes: one group of 32–36 KD forms is noted immediately after the blood meal, followed by the principal forms with Mr around 30 KD about 10 h later; synthesis is terminated at about 24 h after blood meal. Similar results were obtained after in vitro translation of mRNA. Trypsin precursor and trypsin mRNA were not detected by our assay of midguts of unfed females. Trypsin synthesis is induced in a dose-dependent manner by injection of either blood or sugar solutions into isolated midguts. It is concluded that the stimulus for initial trypsin synthesis is mechanical and/or osmotic stress acting independently of the nervous system. Processing of trypsin in the midgut cells involves cleavage of putative signal peptides: in vitro translation in the presence of microsomes led to a constant shift in molecular weights of 1–2 KD prior to secretion. Exposure of washed midgut epithelia from different stages to native blood in vitro inhibited final processing of the intracellular trypsin to the extracellular forms while stimulation of protein synthesis was observed. Consequently the role of the peritrophic membrane in compartmentalization of the digestive process is further emphasized.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

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.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Egg development in the mosquito Anopheles albimanus

Summary

In the mosquito, Anopheles albimanus, previtellogenic egg development was completed by 48 h after emergence, and vitellogenic growth was completed by 36 h after a blood meal. Ecdysteroid levels reached a peak of 800 pg/female by 18 h, while vitellin levels rose to their maximum 36–48 h after a blood meal. Most of the ecdysteroids present in the female before 36 h behaved as ‘free’ hormone, while after 42 h the ecdysteroids were ‘conjugated’. Injection of 20-hydroxyecdysone into non-blood-fed females induced degeneration of the resting stage oocytes, but vitellogenin synthesis was detectable by autoradiography. Injection of 5 μ of 20-hy-hroxyecdysone into blood-fed decapitated females induced almost precisely normal levels of vitellin. Detailed analysis of the effect of decapitating blood-fed females suggested that the release of factors from the head (e.g., egg development neurosecretory hormone) occurs as an all-or-none phenomenon, and probably occurs twice.     

Post-feeding induction of trypsin in the midgut of Aedes aegypti L. (Diptera: Culicidae) is separable into two cellular phases

The induction of trypsin activity in the midgut of the mosquito, Aedes aegypti, was studied following meals of chicken blood, and several protein and peptide diets. Various concentrations of bovine serum albumin (BSA) in 0.15 M NaCl stimulated trypsin activity, in a similar fashion to the initial increase observed after a normal blood meal. Trypsin synthesis was also initiated when Ae. aegypti were fed on glutaraldehyde cross-linked BSA and on BSA fragments prepared by both pepsin and cyanogen bromide cleavage. Non-soluble proteins, in the form of glutaraldehyde-fixed erythrocyte ghosts, induced a delayed and reduced trypsin response, whilst small peptides from neutralized liver digests did not induce trypsin activity until 8–10 h after feeding. Metabolic inhibitors had varying effects on the post-feeding activity of trypsin stimulated by BSA feeding. Cycloheximide, a peptidyl transferase inhibitor prevented expression of all activity in vivo, whereas α-amanitin (RNA-polymerase inhibitor) did not affect trypsin activity in the first 10 h after feeding. At 20 μg/ml concentration in the diet, actinomycin D (RNA synthesis inhibitor) caused temporary superinduction followed by inhibition of trypsin activity, but at lower concentrations, the later phase of trypsin activity was inhibited. The results suggest that post-feeding induction of trypsin activity in Ae. aegypti is a two-phase process regulated at the midgut cellular level. The first phase of trypsin synthesis is stimulated by soluble proteins of variable molecular weights, and only involves translation of messenger RNA already available within the midgut cells. The second phase is stimulated by small peptides and requires complete synthesis of new mRNA from DNA.     

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.     

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.     

Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development

Light microscopy of native preparations, histology, and electron microscopy have revealed that Phlebotomus duboscqi belongs to a class of sand fly species with prompt development of the peritrophic matrix (PM). Secretion of electron-lucent fibrils, presumably chitin, starts immediately after the ingestion of a blood meal and, about 6 h later, is followed by secretion of amorphous electron-dense components, presumably proteins and glycoproteins. The PM matures in less than 12 h and consists of a thin laminar outer layer and a thick amorphous inner layer. No differences have been found in the timing of the disintegration of the PM in females infected with Leishmania major. In both groups of females (infected and uninfected), the disintegration of the PM is initiated at the posterior end. Although parasites are present at high densities in the anterior part of the blood meal bolus, they escape from the PM at the posterior end only. These results suggest that L. major chitinase does not have an important role in parasite escape from the PM. Promastigotes remain in the intraperitrophic space until the PM is broken down by sand-fly-derived chitinases and only then migrate anteriorly. Disintegration of the PM occurs simultaneously with the morphological transformation of parasites from procyclic forms to long nectomonads. A novel role is ascribed to the anterior plug, a component of the PM secreted by the thoracic midgut; this plug functions as a temporary barrier to stop the forward migration of nectomonads to the thoracic midgut. This work was supported by the Ministry of Education of the Czech Republic (projects MSM0021620828 and LC06009).     

Ultrastructural features of the midgut epithelium of females Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae).

A morphological study of the midgut of Lutzomyia intermedia, the primary vector of cutaneous leishmaniasis, in southeast Brazil, was conducted by light, scanning and transmission electron microscopy. The midgut is formed by a layer of epithelium of columnar cells on a non-cellular basal lamina, under which there is a musculature, which consists of circular and longitudinal muscular fibers. A tracheolar network is observed surrounding and penetrating in the musculature. Females were examined 12, 24, 48, 72 h and 5 days following a blood meal and were analyzed comparatively by transmission electron microscopy with starved females. In starved females, the epithelium of both the anterior and posterior sections of the midgut present whorl shaped rough endoplasmic reticulum. The posterior section does not present well-developed cellular structures such as mitochondria. Observations performed at 12, 24, 48 and 72 h after the blood meal showed morphological changes in the cellular structures in this section, and the presence of the peritrophic matrix up to 48 h after the blood meal. Digestion is almost complete and a few residues are detected in the lumen 72 h after blood feeding. Finally, on the 5th day after the blood meal all cellular structures present the original feature resembling that seen in starved sand flies. Morphometric data confirmed the morphological observations. Mitochondria, nuclei and microvilli of midgut epithelial cells are different in starved and blood fed females. The mitochondria present a similar profile in the epithelium of both the anterior and posterior section of the midgut, with higher dimension in starved females. The cell microvilli in the posterior section of the midgut of starved females are twice the size of those that had taken a blood meal. We concluded that there are changes in the midgut cellular structures of L. intermedia during the digestion of blood, which are in agreement with those described for other hematophagous diptera.