Proteolytic enzymes of female Anopheles: Biphasic synthesis,regulation, and multiple feeding

Two types of trypsin activity were detected in Anopheles albimanus, a constitutive and an inductive component, which have identical immunopatterns. The constitutive trypsin in synthesized shortly after eclosion and is retained in the midgut epithelial cells. The inductive trypsin is synthesized and released continuously after a blood meal has been ingested; maximal activities vary between 12 h and 18 h after a blood meal. Once digestion is completed, trypsin is excreted, but the constitutive trypsin level is restored within 24 h, before the next blood meal is taken. In A. gambiae, A. Stephensi, and A. quadrimaculatus, the constitutive trypsin component is also present, but at much lower levels. In A. albimanus fed multiple blood meals at 24 h intervals, trypsin oscillates at nearly maximal levels as long as blood is present in the midgut and depending on the ovarian status. Expression of the two trypsin components in A. albimanus was found to be independent of the neurosecretory system, but synthesis of the constitutive trypsin appears to require the presence of the corpora allata. In all species tested, chymotrypsin is secreted after a blood meal in a similar temporal pattern as trypsin, but it is never present before the blood meal. Reinvestigating several aedine species for the presence of chymotrypsin by using different substrates revealed measurable quantities in blood-fed females compared to earlier reports. Equally, aminopeptidase activity is present in all species tested and characterized by a constitutive component. Its activities follow different temporal patterns than the endopeptidases. © 1995 Wiley-Liss, Inc.     

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.     

FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.     

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.     

Histochemical changes in the midgut of two ixodid tick speciesBoophilus microplus andRhipicephalus appendiculatus during digestion of the blood meal

The changes in the midgut epithelia of two ixodid tick species,Boophilus microplus andRhipicephalus appendiculatus, have been studied using several histochemical techniques. It was revealed that there is an accumulation of RNA at the time of tick attachment to the host and prior to the arrival of the blood meal, indicating that the midgut digest cell is furnished with the machinery characteristic of a synthetic cell. There appears to be a synchrony in the appearance of granules with peroxidase activity and the uptake of haemoglobin into the midgut digest cells. Alkaline phosphatase activity was observed in the midgut epithelia of all ticks except in a few of the long-starved ticks, and was concentrated in the apical plasma membrane regions of those digest cells involved in absorption and the intracellular digestion of haemoglobin. The presence of these enzymes suggests that the midgut digest cell is a multifunctional cell capable of both secretory and digestive activities. The colloidal material in the midgut lumen was found to result from the accretion of several products both secreted and excreted by the midgut epithelial cells and exhibited different staining reactions depending on which component dominated. The nature of the material suggests that in addition to its digestive function it may serve as a sink to bind all the by-products of digestion and thereby facilitate their excretion.     

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 secretory dynamics of the CHH-producing cell group in the eyestalk of the crayfish,Astacus leptodactylus,in the course of the day/night cycle

Summary The secretory dynamics of the Crustacean Hyperglycemic Hormone (CHH)-producing cells in the eyestalk of the crayfish Astacus leptodactylus were studied during the daily cycle (12 h light/12 h dark). The different secretory stages of individual cells were determined by means of immunocytochemistry combined with morphometric analysis at the light-microscopic level. The data obtained were correlated with the 24-h rhythmicity of blood glucose concentration. The results suggest the following hypothesis. The synthetic activity of the CHH cells receives a stimulus 2 h before the beginning of the dark period, resulting in a pronounced transfer of CHH granules into the axons. These CHH granules reach the axon terminals after the onset of the dark period. At that time a burst of exocytotic activity occurs, causing a strong release of CHH into the hemolymph. Four hours later this CHH release results in hyperglycemia. The same process, though with less intensity, is repeated and causes a second smaller glucose peak at the beginning of the light period.     

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.     

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.     

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.     

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.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

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.     

The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species.

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.     

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.     

INDUCTION OF ENDOMETRIAL PEROXIDASE SYNTHESIS AND SECRETION BY ESTROGEN AND ESTROGEN ANTAGONIST

Synthesis of peroxidase was induced in the uterine epithelium of immature rats by multiple doses over a 24–96-h period of either 17 β-estradiol, the estrogen-antagonist Parke-Davis CI-628, or a combination of estradiol plus antagonist. Endogenous peroxidase activity first appeared in the cisternae of the rough endoplasmic reticulum of surface epithelial and glandular cells within 24–48 after the initial injection. Uterine peroxidase activity was also visible in the cisternae of the Golgi apparatus, in Golgi-derived secretory granules, and within the uterine and glandular lumen. Some cells of the epithelium produced little or no peroxidase, even after 96 h. Whereas the antagonist appeared to induce synthesis and secretion of peroxidase, neither the antagonist alone nor the combined treatment (estradiol plus antagonist) reproduced the estradiol-mediated growth in organ size and increased lumen diameter.     

Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae

Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.