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Journal of Insect Physiology 51 (2005) 769–776

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Morphological and enzymatic analysis of the midgut ofAnopheles darlingi during blood digestion

Kendi Okudaa, Abrahim Carocia, Paulo Ribollab, Osvaldo Marinottic,Antonio G. de Bianchia, A. Tania Bijovskya,�

aDepartamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, CEP 05508-900, BrazilbDepartamento de Parasitologia, Instituto de Biociencias, Universidade Estadual Paulista, Botucatu, SP, BrazilcDepartment of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA 92697, USA

Received 20 December 2004; received in revised form 9 March 2005; accepted 14 March 2005

Abstract

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered

epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each

cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic

reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei.

Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3 h post bloodmeal (PBM), nucleoli

enlarge manyfold. An intense absorptive process takes place during the first 24 h PBM, with the formation of large glycogen

inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their

maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with

their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested

erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive

process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of

neutralize its toxicity.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Anopheles darlingi; Midgut; Mosquito; Ultrastructure; Protease

1. Introduction

Adult insects live in a wide range of habitats anddisplay enormous variation in appearance, life style anddiet. Haematophagy, the habit of blood feeding, evolvedindependently several times among insects, and multiplebehavioural, morphological, and physiological adapta-tions accompanied this evolutionary trend. There isample evidence that the host-seeking behaviour ofhaematophagous insects is mediated by semiochemicals

e front matter r 2005 Elsevier Ltd. All rights reserved.

sphys.2005.03.010

ing author. Tel.: +5511 3091 7404;

1 7417.

ess: bijovsky@icb.usp.br (A.T. Bijovsky).

emanating from their hosts (Justice et al., 2003), thattheir proboscis are adapted for cutting or piercing thetissues of a host (Clements, 1992), that their salivaryglands secret products such as anti-clotting, anti-plateletand vasodilatory compounds that antagonize vertebratehaemostasis (James, 2003), and that their midguts arewell adapted to respond quickly secreting a wide varietyof digestive enzymes following a bloodmeal (Noriegaand Wells, 1999; Okuda et al., 2002). Because pathogensare transmitted to humans due to haematophagy, thestudies of mechanisms and adaptations that insectsevolved to enable and succeed in finding, ingesting anddigesting a bloodmeal have been extensively investigatedto find novel ways to disrupt or diminish pathogen

ARTICLE IN PRESSK. Okuda et al. / Journal of Insect Physiology 51 (2005) 769–776770

transmission. Based on this premise, anopheline mos-quitoes, vectors of human malaria, have been the objectof extensive investigation. Morphological and func-tional aspects of their midguts have been intensivelystudied (Hecker, 1977; Clements, 1992; Siden-Kiamosand Louis, 2004; Abraham and Jacobs-Lorena, 2004),focusing mainly on the role of digestive enzymes,microvillar proteins and the peritrophic membrane infood digestion and parasite development. However,differences in midgut physiology that account forvarious factors affecting blood digestion and parasitedevelopment have been observed among mosquitospecies (Beier, 1998). Temporal differences exist in thekinetics of proteases synthesis and secretion (Caroci etal., 2003) and peritrophic membrane formation (Pon-nudurai et al., 1988). These studies show that eachmalaria vector has a characteristic time frame in whichblood is digested and malaria parasites need to developinto oocysts before they are destroyed by digestiveenzymes. Despite the importance of the midgut inmosquito survival, reproduction and parasite transmis-sion, only a small number of studies have beenconducted to understand the digestive process inneotropical anophelines (Chadee and Beier, 1995; deAlmeida et al., 2003; Caroci et al., 2003). In this work,we describe our initial investigations on the morphologyand biochemical components of the midgut of An.

darlingi, a major human malaria vector in SouthAmerica. Our observations are similar but not identicalto those previously described for other mosquitoes.

2. Material and methods

2.1. Animals

Adult females of An. darlingi were collected in PortoVelho, Rondonia (81490S, 631540W), manually usinghumans as baits. The taxonomic identification was madein accordance with Faran (1980) and Faran andLinthicum (1981). The captured females were main-tained in an insectary for oviposition and only the F1progeny was used in the experiments.

After hatching, larvae were fed with ground fish food(TetraMin, TetraWerke, Germany). Pupae were trans-ferred to distilled water and placed in cages until theemergence of adults. Adults were fed ad libitum on 10%sucrose solution and kept at 25 1C, 75% relativehumidity, and photoperiod of 12 h dark–12 h light.When needed, 4-day-old adult female mosquitoes werefed on anaesthetised Balb/c mice (0.3mg/kg of 2-(2,6-xylidino)-5,6-dihydro-4H-1,3-thiazine hydrochloride(Rompun, Bayer) plus 30 mg/kg acepromazin (Acepran,Univet S.A., Sao Paulo, Brazil) and only those fullyengorged were used for the experiments.

In order to better visualize the participation of midgutregions in the digestive process, females were fed onsucrose solution coloured with 5% blue alimentary dye(Arco-ıris, Brazil). The blood and sugar fed females werecarefully dissected and the alimentary tract was photo-graphed under a stereoscopic microscope.

2.2. Preparation of samples for electron microscopy

Digestive tracts were dissected in PBS and theblood content was removed when necessary. Tractswere fixed for 90min in 6% glutaraldehyde in sodiumcacodylate buffer (pH 7.2), at room temperature.After postfixation with 2% osmium tetroxide, pieceswere contrasted en bloc with 2% uranyl acetate in70% acetone and dehydrated in acetone. In order toobserve the same area, midguts were always trimmedup to reach the central portion of the posterior regionsbefore the embedding in Spurr’s resin (modified fromHecker et al., 1974). Semithin sections (200–300 nm)were stained with toluidine blue for light microscopystudies. Ultrathin sections (60–70 nm) of each region,stained with uranyl acetate and lead citrate, wereobserved at 80 kV in a JEOL 100CX transmissionelectron microscope.

2.3. Preparation of samples for biochemical analysis

Midguts from five blood fed females were dissected inPBS (130mM NaCl, 7mM Na2HPO4, 10mM NaH2-

PO4), pH 7.2 and transferred to Eppendorf tubescontaining 0.5ml of double distilled water. The collectedmidguts were homogenised, centrifuged at 10,000� g

for 10min, at 4 1C, and the supernatants were stored at�20 1C until use. Three samples were prepared for eachexperimental time point; non-blood fed mosquitoes, 0,3, 6, 12, 18, 24, 36, 48, 60 and 72 h post bloodmeal(PBM).

2.4. Determination of hydrolase activity

Chymotrypsin activity was assayed using the chromo-genic substrate N-succinyl Ala–Ala–Pro–Phe p-nitroa-nilide (AAPF, Sigma Chemical, St. Louis, USA) andaminopeptidase activity was tested with Ala p-nitroani-lide, Arg p-nitroanilide, Leu p-nitroanilide, and Met p-nitroanilide (Sigma Chemical, St. Louis, USA). Briefly,substrates were dissolved to 10mM in dimethyl sulf-oxide and diluted to 1mM final concentration in PBS,pH 8.0 immediately prior to each assay. Midguthomogenates (4 ml for chymotrypsin and 10 ml foraminopeptidase determinations) and substrate solutions(200 ml) were mixed and optical density, at 405 nm, wascontinuously monitored at 30 1C for 1 h. Enzymeactivity was calculated using an extinction coefficientof 8800mM/cm (Erlanger et al., 1961). One enzyme unit

ARTICLE IN PRESSK. Okuda et al. / Journal of Insect Physiology 51 (2005) 769–776 771

is defined as the activity required to hydrolyse 1 mmol ofsubstrate per min. Mouse blood processed under thesame conditions was used as control. Experiments wereperformed in ELISA plates using an iEMS Analysermicroplate reader (Labsystems Oy, Finland).

Fig. 2. Posterior midgut of non-blood fed female of An. darlingi. The

midgut epithelium is composed by digestive cells that present an

intricate basal labyrinth (B) at their basolateral membrane, and

3. Results

Adult female mosquitoes feed both on sucrosesolution and blood. However the destination of theingested food varies according to the diet composition.While the ingested sucrose solution is stored in thediverticulum and distributed along both midgut regions(Fig. 1a) the bloodmeal is conducted exclusively to theposterior midgut (Fig. 1b). Therefore, our studies weredirected to the posterior region of the mosquito midgut,specifically to the processes involved in blood digestion.The posterior midgut epithelium is a single layercomposed mostly of digestive, cuboidal cells. Somebasal cells, presumably regenerative and endocrinecells, are dispersed between the abundant digestive cells(Fig. 2). A thick, multilayered basal lamina supports theepithelium, which is involved in a network of tracheolesand longitudinal and circular muscle bundles. Thedigestive cells present, at their basolateral membrane,an intricate basal labyrinth and a belt of intercellularjunctions (mainly zonula continua) joins their micro-villated apical poles. Their nuclei are centrally located

Fig. 1. Spatial distribution of ingested meals in An. darlingi alimentary

canal. (a) The blue coloured sucrose solution is stored in the ventral

diverticulum (D) and distributed along anterior (A) and posterior (P)

midgut. (b) The ingested blood is confined to the posterior midgut.

MT: Malpighian tubules. Bar: 0.5mm.

numerous microvilli (Mv) at the apical pole. Their nuclei (N) are

centrally located with scarce heterochromatin. Profiles of rough

endoplasmic reticulum (R), sometimes with whorled outlines, are

neighbouring the nuclei. Numerous round granules (arrow) fill the

apical cytoplasm. Bar: 1mm.

with an evident nucleolus and scarce heterochromatin.Profiles of rough endoplasmic reticulum, sometimeswith whorled outlines, neighbours the nucleus. Beforethe blood engorgement, apical cytoplasm is filled withnumerous round granules (Fig. 2).

Several simultaneous events take place almost im-mediately after blood feeding. Engorgement causes agreat distension of the midgut which, in turn, provokesthe flattening of digestive cells. Their height, approxi-mately 14 mm before blood feeding, change to 4.8 mm,with the concomitant flattening of their nuclei. Otherstriking events that occur immediately after engorge-ment are the disappearance of the apical granules,and the disassembling of the whorls of endoplasmicreticulum.

Changes in the morphology of the midgut continueand at 3 h PBM the nucleolus of digestive cells appearsmuch enlarged (Fig. 3). At 18 h PBM, an observedreduction in the alimentary bolus volume allows the

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Fig. 3. Posterior midgut of An. darlingi females at 3 h post bloodmeal.

The epithelial cells are stretched, Nucleolus (Nu) is quite enlarged. The

whorls of rough endoplasmic reticulum and the apical granules

disappeared. Lu: lumen; N: nucleus. Bar: 1 mm.

Fig. 4. Posterior midgut of An. darlingi females at 18 h post blood-

meal. Electrondense aggregates (Ag) above the peritrophic membrane

seem to derive from erythrocyte (E) lysis. The digestive cells contain

large glycogen inclusions (G). B: basal labyrinth; Mv: microvilli. Bar:

1mm.

Fig. 5. Posterior midgut of An. darlingi females at 36 h post blood-

meal. Luminal aggregates form a multilayered structure (Ag) on the

peritrophic matrix (PM). The apical granules reappear (arrows).

B: basal labyrinth; M: mitochondria; Mv: microvilli. Bar: 1 mm.

K. Okuda et al. / Journal of Insect Physiology 51 (2005) 769–776772

midgut epithelium to begin returning to its previousheight. A large number of rough endoplasmic reticulumprofiles and many vesicles and large glycogen inclusionsare found in the cytoplasm of digestive cells at this time.Granules begin to reappear at the cellular apical polearound 24 h PBM.

Beginning at 18 h PBM, blood digestion is visuallyevident with the presence of lysed erythrocytes in theperiphery of the alimentary bolus (Fig. 4). The digestionof erythrocytes causes a gradual deposition of coarseelectrondense aggregates on the luminal face of thealready thick peritrophic membrane. This coarse mate-rial persists during all the digestive process, forming amultilayered structure, which is excreted at the end ofthe digestive process (Fig. 5).

At the end of blood digestion process, around 60 hPBM, the morphology of the epithelium returns to thecharacteristics observed before the engorgement, withthe reassembling of the whorls of endoplasmic reticu-lum. However, several glycogen inclusions as well aslipid inclusions are still present in the basal region ofdigestive cells.

Changes in proteolytic activity accompany the mor-phological transformations previously described. Chy-motrypsin, trypsin, and aminopeptidase activities areinduced by a bloodmeal. Chymotrypsin and trypsin

ARTICLE IN PRESSK. Okuda et al. / Journal of Insect Physiology 51 (2005) 769–776 773

activities are induced as soon as 3 h PBM, attainingmaximum values (�60 and �85mU/midgut, respec-tively) at 10–20 h PBM. The activity remains unaltereduntil 36 h PBM, decreasing afterward to levels thatare higher than those of non-blood fed mosquitoes(Fig. 6). Aminopeptidase activity also is induced bya bloodmeal. Enzymatic activities measured with Alap-nitroanilide (Fig. 7) and with Arg p-nitroanilide(not shown) have similar patterns and give a maximumat 48 h PBM while the activities measured with Leup-nitroanilide (Fig. 7) and with Met p-nitroanilide (not

0

20

40

60

80

100

120

0 12 24 36 48 60 72

mU

/mid

gut

Chymotrypsin Trypsin

Time after blood feeding (h)

Fig. 6. Hydrolytic activities of An. darlingi midgut extracts. Three

samples were assayed for each experimental time point and the results

are represented as mean7SD. Variations in trypsin and chymotrypsin

activities in the midguts of mosquitoes as a function of time after

ingestion of a bloodmeal. Enzyme activities were determined by

hydrolysis of artificial substrates (chymotrypsin, N-succinyl Ala–

Ala–Pro–Phe p-nitroanilide and trypsin, Benzoyl-DL-arginine-p-nitroa-

nilide). The data for trypsin activity were compiled from Caroci et al.

(2003).

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48 60 72

mU

/mid

gut

LeupNA

Time after blood feeding (h)

AlapNA

Fig. 7. Hydrolytic activities of An. darlingi midgut extracts. Three

samples were assayed for each experimental time point and the results

are represented as mean7SD. Variations in aminopeptidase activity in

the midguts of mosquitoes as a function of time after ingestion of a

bloodmeal. Enzyme activities were determined by hydrolysis of

artificial substrates (Ala p-nitroanilide and Leu p-nitroanilide).

shown) show a different pattern with maximum value at36 h PBM.

4. Discussion

The posterior midgut is the site of bloodmealdigestion and absorption in mosquitoes (Lehane,1991). Accordingly, blood ingested by Anopheles darlingi

adult females is directed to that region of the midgut(Fig. 1b) where it remains for the entire digestiveprocess. However, sugar meals are directed to thediverticula (Fig. 1a) where digestion is initiated bysalivary carbohydrases (Schaefer and Miura, 1972;Marinotti et al., 1996). The sugar meal stored in thediverticula is slowly released into the midgut wheredigestion is completed and absorption occurs (Clements,1992). However, An. darlingi apparently store the sugarmeal in the diverticula for a short time. Less than 1% ofthe wild caught An. darlingi females had sugar meal intheir diverticula (Pajot et al., 1975).

The time taken for mosquitoes (Anopheles, Aedes andCulex) to digest a bloodmeal is approximately 60–70 h,but it is strongly influenced by ambient temperature,blood source, meal size, and several other factors(Lehane, 1991). Our observations indicated that, at25 1C, An. darlingi completed the digestive process inapproximately 60 h. Morphological and biochemicalmodifications that occur during the progression ofblood digestion were investigated.

The ultrastructure of An. darlingi female midguts(posterior region) resembles that described for othermosquitoes and it is consistent with the functionsattributed to the organ, i.e., synthesis and secretion ofperitrophic membrane components and of digestiveenzymes and absorption of the products of the blooddigestion (Hecker, 1977; Okuda et al., 2002).

As described for other anophelines, in An. darlingi theengorgement and the consequent midgut distensionseem to trigger the secretion of apical granules storedin the digestive cells (Figs. 2 and 3). These granulescontain peritrophic membrane precursors and digestiveenzymes (Freyvogel and Staeubli, 1965; Berner et al.,1983; Devenport et al., 2004). Additionally, afterengorgement, we observed the disassembly of the whorlsof endoplasmic reticulum and the enlargement ofnucleolus of digestive cells, as shown in Fig. 3. Thesemorphological changes are associated with the commit-ment of these cells to synthesize digestive enzymes.Ingestion of blood provokes, in Ae. aegypti, thedispersion of rough endoplasmic reticulum whorls(Berner et al., 1983) and induces proteolytic activity inAn. stephensi midgut (Perrone and Spielman, 1988).

The peritrophic membrane is fully formed in anophe-line mosquitoes between 24 and 48 h PBM, while thefirst signs of this structure is observed as soon as 12 h

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PBM (Ponnudurai et al., 1988). As can be seen inFigs. 4 and 5, in An. darlingi, we observed a similartime of peritrophic membrane formation. Peritrophicmembrane has been already described as a protectionagainst the sharp edges of haemoglobin-like crystalsin the gut of An. stephensi (Berner et al., 1983) andas a mechanical barrier for pathogenic organisms suchas viruses, bacteria, and also parasites, includingPlasmodium sp. (Billingsley and Rudin, 1992; Shaoet al., 2001).

Erythrocyte lysis is concurrent with the deposition ofelectrondense aggregates at the luminal face of theperitrophic membrane, observed in Fig. 4, at 18 h PBM.Probably, these electrondense structures contain inso-luble forms of haem. Mosquitoes, as well as otherhaematophagous insects, have developed several me-chanisms to counteract the oxidative challenge due tohaem release following digestion of haemoglobin bymidgut proteinases. The bug Rhodnius prolixus, forexample, transforms haem to haemozoin, an insolublecomponent that forms large and electrondense aggre-gates in the gut lumen (Oliveira et al., 1999, 2000). Ae.

aegypti in its turn neutralises most of the haem, bindingit to peritrophic membrane (Pascoa et al., 2002) and,synthesizes ferritin as a cytotoxic protector (Geiser et al.,2003).

Reappearance of apical granules in the digestive cells(here illustrated in Fig. 5, at 36 h PBM) is observed from24 h PBM, suggesting that the insect began to preparefor a new blood intake even before finishing thedigestion of the previous meal. Accordingly, studieshave shown that anophelines may take multiple blood-meals in a single gonotrophic cycle (Briegel and Horler,1993; Koella et al., 1998; Nirmala et al., 2005).Controversial data, however, indicate that the require-ment of more than one bloodmeal to complete agonotrophic cycle is infrequent among field-collectedAn. darlingi (Lounibos et al., 1998). Possibly, theoccurrence of multiple bloodmeals is determined byboth genetic (species) and environmental factors.Food availability during larval development, for in-stance, determines the size of adult mosquitoes andconsequently the volume of blood ingested at eachbloodmeal.

Storage inclusions (lipid droplets and glycogen inclu-sions, Fig. 4) are formed in the digestive cells during thefirst two days PBM and disappear only after 72 h PBM.Possibly, the transport of some digestion products(lipids and carbohydrates) to haemolymph does notoccur at the same rate as the digestive process.Alternatively, since blood is composed of mostlyprotein, the storage inclusions may contain the resultof metabolic conversion of amino acids into carbohy-drates and lipids.

As observed for other mosquitoes, proteolytic activ-ities increased in the midguts of An. darlingi, following a

bloodmeal. As illustrated in Fig. 6, chymotrypsinand trypsin activities increase during the initial 24 hPBM, reaching a plateau (�60 and �85mU/midgut,respectively) that is maintained at least until 36 hPBM. The activities decrease gradually from then on.The measured activities however, represent the com-bined actions of at least two An. darlingi digestivetrypsins (Caroci et al., 2003) and possibly two chymo-trypsins (de Almeida et al., 2003) previously character-ized. Vizioli et al. (2001) showed that the transcriptionof two An. gambiae chymotrypsin genes, Anchym1 andAnchym2, is induced by bloodmeal while two trypsinsare induced in the midguts of An. gambiae (Muller et al.,1995), An. albimanus (Horler and Briegel, 1995), An.

aquasalis and An. albitarsis (Caroci et al., 2003). Thesedifferent enzymes probably perform complementarydigestive functions. Supporting this hypothesis, Mulleret al. (1993) showed that while the An. gambiae trypsin 1(Antryp 1) has no preference for albumin overhaemoglobin, Antryp 2 hydrolyses haemoglobin morereadily.

Two different patterns of aminopeptidase activitywere observed in the midgut extracts of An. darlingi as isshown in Fig. 7. Activities toward Ala p-nitroanilide andArg p-nitroanilide are induced 3 h PBM, increase duringthe first 12 h PBM and remain constant thereafter until36 h PBM (�60mU/midgut). A second peak of activityoccurs later during the digestive process, at 48 h PBM(raising the activity to 90mU/midgut), followed bygradual decrease. Hydrolysis of Leu p-nitroanilide andMet p-nitroanilide follows a different pattern, increasinggradually from 3h PBM until 36 h PBM and attaining amaximum of �40mU/midgut. The activity decreasesafterwards reaching levels identical to those of non-blood fed mosquitoes at 60 h PBM. The identifiedsubstrate specificities and temporal expression patternsindicate the occurrence of at least two distinct digestiveaminopeptidases in An. darlingi. At least three amino-peptidases are responsible for the digestion of proteinsin An. stephensi (Billingsley, 1990).

This is the first morphological description of themidgut of a neotropical anopheline (subgenus Nyssor-hynchus). Furthermore, this study was conductedwith Anopheles darlingi, a major human malaria vectorin South and Central America (Tadei and DutaryThatcher, 2000). The information presented here in-creases our knowledge of basic mosquito biology andcan serve as basis for future studies of malaria parasiteinteractions.

Acknowledgments

This work was supported by grants from Fundac- aode Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) and Conselho Nacional de Desenvolvimento

ARTICLE IN PRESSK. Okuda et al. / Journal of Insect Physiology 51 (2005) 769–776 775

Cientıfico e Tecnologico (CNPq). K.O. and A.C. weregrantees of graduate FAPESP fellowship. A.G.B. is aCNPq research fellow.

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