Fatty acid incorporation by Rhodnius prolixus midgut

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.     

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.     

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.     

Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase

The utilization of dietary lipids was studied in adult females of Rhodnius prolixus with the use of radiolabeled triacylglycerol (TAG). It was shown that (3)H-triolein, when added to blood meal, was hydrolyzed to free fatty acids in the posterior midgut lumen. Subsequently, free fatty acids were absorbed by posterior midgut epithelium and used in the synthesis of phospholipids, diacylglycerol (DAG) and TAG. Phospholipids, DAG and free fatty acids were then found in hemolymph, from where they were rapidly cleared, and label was found in the fat body, mainly associated with TAG. Radioactive lipids, especially TAG and phospholipids, also accumulated in the ovaries. The TAG-lipase activities of posterior midgut luminal content and tissue were characterized by incubation of these samples with (3)H-triolein in the presence of the detergent Triton X-100 and determination of the amounts of released radioactive free fatty acids. Under the conditions employed here, the release of free fatty acids was proportional to the incubation time and to the amount of sample obtained from insect midgut (enzyme source) that was added. TAG-lipase activities were affected by pH and posterior midgut tissue showed optimum activity around pH 7.0-7.5, but the luminal content had the highest activities as pH decreased. Differences in activities were observed according to calcium concentration in the medium. TAG-lipase activities were also affected by the concentration of NaCl and were activated in the presence of increasing salt concentrations. These activities were inhibited by phenylmethylsulphonyl fluoride (PMSF). On the second day after blood meal, when digestion is very intense, TAG-lipase activities were maximal and then gradually decreased.     

Internal regulation of rate of digestion of blood meals in the mosquito, Aedes aegypti.

Rate of digestion of blood meals proceeded more rapidly in females of Aedes aegypti that were inseminated or injected with matrone (extract of male accessory glands) than in virgin females. Digestion rate was determined by interfacial precipitin tests and immunodiffusion tests for undigested blood-meal proteins. Application of a cervical ligature within 1 hr of blood feeding retarded the digestion rate. Ligated females that received brain transplants from blood-fed donors digested their blood meals at a rate similar to that of unligated controls. When ligatures were not applied until 12 hr after feeding no delay in digestion was observed.     

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.     

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.     

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.     

Ultrastructure of the midgut and blood meal digestion in the adult tickDermacentor variabilis

Digestive cells in the midgut of male and femaleDermacentor variabilis (Say) took up the blood meal in coated vesicles and smooth flask-shaped vesicles, and deposited it in endosomes which were digested via heterophagy. Iron was concentrated in residual bodies.Digestion occurred in three distinct phases in mated females: (1) continuous digestion (initiated by feeding) occurred during slow engorgement; (2) reduced digestion (initiated by mating) occurred in mated females during the period of rapid engorgement; (3) a second continuous digestion phase (initiated by detachment from the host) occurred throughout the post-feeding periods of preoviposition and oviposition.It is proposed that the stem cells in the midguts of unfed females were progenitors of digestive, replacement, and presumed vitellogenic cells in midguts of mated feeding females. Digestive cells were present in all three digestion phases. Only during the first continuous digestion phase did digestive cells fill up with residual bodies, rupture and slough into the lumen, or did whole cells slough into the lumen. During the other two digestion phases no sloughing of digestive cells was observed. At the end of oviposition the digestive cells were filled with residual bodies. Replacement cells were present only during the first continuous-digestion phase. Presumed vitellogenic cells were present only during the reduced-digestion phase and during the second continuous-digestion phase. Stem cells in unfed males developed only into digestive cells in feeding males. Fed males and fed unmated females had only the first continuous-digestion phase. After being hand-detached from the host, unmated 13-day-fed females went through cellular changes associated with the reduced-digestion phase and second continuous-digestion phase of fed mated females, then began ovipositing. Maximum development of the basal labyrinth system and lateral spaces matched the known time of maximum water and ion movement across the midgut epithelia.Spectrophotometric analyses of lumen contents and midgut cells, sampled after detachment from the host, showed that concentrations of protein and hemoglobin at day 1 post-detachment decreased by one-half at the beginning of oviposition, while hematin increased about twofold by the end of oviposition. This supported the idea of the presence of a second continuous-digestion phase.     

The digestion of dandelion pollen by adult worker honeybees

ABSTRACT. The digestion of dandelion ( Taraxacum officinale ) pollen by adult worker honeybees ( Apis mellifera Linn.; Hymenoptera: Apidae) was initiated at the germination pores. 30 min after feeding, pollen had reached the anterior midgut and the germination pores had become swollen. This permitted further removal of protoplasm during the next 2 h of digestion as the pollen passed into the middle portion of the midgut. 3 h after feeding, pollen grains had reached the posterior midgut where some had ruptured to release both 'naked' protoplasm and masses of protoplasm but many remained intact or were only partially digested; undigested pollen grains passed unchanged to the rectum. The lipid-rich pollenkitt layer was removed from the exine during digestion. Our studies indicate that dandelion pollen was not utilized completely by honeybees.     

Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

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.     

Influence of the endocrine system on tryptic activity in female Aedes aegypti

In MNC-ablated or ovariectomized mosquitoes, blood-fed by enema, the tryptic activity of midgut homogenates was reduced by half. By sealing the anus after the blood meal, we showed that the reduction in enzyme activity was not due to premature or increased excretion of active enzyme in operated females. The enzyme activity was restored to the level of unoperated controls by either reimplantation of an ovary or injection of 20-hydroxy-ecdysone in a large, single dose shortly before the blood meal.The reduction in tryptic activity in MNC-ablated or ovariectomized females did not alter the rate of protein digestion as measured by the decrease of protein in midgut homogenates. We conclude that the neurosecretory system and the ovaries are required for maximal tryptic activity in normal females, and that the enzyme apparently is secreted in excess of that required for complete digestion of the blood meal.