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2792
INTRODUCTION
Lutzomyia longipalpis (Lutz and Neiva 1912) is the main
phlebotomine vector of Leishmania infantum (Nicolle 1908) [syn.
L. chagasi (Cunha and Chagas 1937)], the etiologic agent of the
visceral leishmaniasis in the Americas (Soares and Turco, 2003).
As for any other haemathophagous insects, nutrient digestion and
absorption after blood feeding is one of the most important events
for L. longipalpis, which uses these nutrients to produce eggs.
Since the digestive processes are essentially enzymatic and
enzyme activities are influenced by the hydrogen ion concentration
(pH) in the intestinal environment, studies of pH and the mechanisms
involved in pH control become extremely relevant. This importance
increases if it is considered that Leishmania parasites ingested
through an infective blood meal develop exclusively in the
phlebotomine gut, from where they are transmitted to a vertebrate
host by biting (Bates and Rogers, 2004). In fact, pH could be one
of the most important factors in Leishmania development within
the vector, as well as for the normal functioning of the gut. It has
been shown that Leishmania promastigotes cultivated at pH5.5
differentiate into metacyclic forms in much larger numbers than
those cultivated at pH7.6 (Bates and Tetley, 1993; Zakai et al.,
1998). Acidification of the medium seems to be one of the main
stimuli that determines the differentiation of Leishmania in vitro
and probably plays a similar role during their development in the
sand fly gut (Gontijo et al., 1998).
Although unknown in mammals, a high gut pH (above 9.0) is
common in some insects, especially in larvae from the orders
Lepidoptera and Diptera (suborder Nematocera only) (Terra et al.,
1996). Some models have been proposed in order to explain the
high physiological pH observed in these insects. All of them include
the participation of the enzyme carbonic anhydrase and the hydration
of CO2 molecules (Boudko et al., 2001a; Boudko et al., 2001b). In
adult females of phlebotomine sand flies and mosquitoes
(Nematocera), the physiology of the midgut is different. These
insects go from a diet composed basically of carbohydrates to one
of blood. This implies great modifications in the midgut physiology
especially in the production of digestive enzymes and the promotion
of a slightly alkaline pH necessary for their action.
Studying the pH of the blood bolus inside the midgut of some
blood-fed mosquitoes, Billker and colleagues proposed that the
alkalization observed [pH7.4 to 7.52 and 7.58 in Aedes aegypti
(Linnaeus 1762) and Anopheles stephensi (Liston 1901),
respectively] might be attributed to the phenomenon of CO2
volatilization (Billker et al., 2000). The loss of CO2 from blood
equilibrating with air leads to a reduction in H+ concentration in
accordance with the equation: CO2+H2O}H2CO3}HCO3–+H+.
The authors showed that the pH of 2–3μl of blood reached a value
of 8.0 when exposed to the air for 10min.
On the other hand, del Pilar Corena and colleagues observed a
higher alkalization, to pH8 or more, in the midgut of seven species
of adult mosquito after feeding (del Pilar Corena et al., 2005). Taking
into account the fact that these mosquitoes were fed not with blood
but with a substitute solution containing indicator dyes, the CO2
volatilization mechanism cannot be considered the only mechanism
responsible for the alkalization observed in blood-fed insects.
Probably, both the CO2 volatilization and a second mechanism
The Journal of Experimental Biology 211, 2792-2798
Published by The Company of Biologists 2008
doi:10.1242/jeb.019836
The physiology of the midgut of Lutzomyia longipalpis (Lutz and Neiva 1912): pH in
different physiological conditions and mechanisms involved in its control
Vânia C. Santos, Ricardo N. Araujo, Luciane A. D. Machado, Marcos H. Pereira and Nelder F. Gontijo*
Department of Parasitology, Federal University of Minas Gerais–UFMG, Avenue Antônio Carlos 6627, 31270-901, Belo Horizonte,
MG, Brazil
*Author for correspondence (e-mail: nelder@icb.ufmg.br)
Accepted 25 June 2008
SUMMARY
Nutrient digestion and absorption after blood feeding are important events for Lutzomyia longipalpis, which uses these nutrients
to produce eggs. In this context, the pH inside the digestive tract is an important physiological feature as it can markedly
influence the digestive process as well as interfere with Leishmania development in infected phlebotomines. It was described
previously that unfed females have an acidic midgut (pH6). In this study, the pH inside the midgut of blood-fed females was
measured. The abdominal midgut (AM) pH varied from 8.15±0.31 in the first 10h post-blood meal to 7.7±0.17 after 24h. While the
AM was alkaline during blood digestion, the pH in the thoracic midgut (TM) remained acidic (5.5–6.0). In agreement with these
findings, the enzyme α-glucosidase, which has an optimum pH of 5.8, is mainly encountered in the acidic TM. The capacity of
unfed females to maintain the acidic intestinal pH was also evaluated. Our results showed the presence of an efficient mechanism
that maintains the pH almost constant at about 6 in the midgut, but not in the crop. This mechanism is promptly interrupted in the
AM by blood ingestion. RT-PCR results indicated the presence of carbonic anhydrase in the midgut cells, which apparently is
required to maintain the pH at 6 in the midgut of unfed females. Investigations on the phenomenon of alkalization observed after
blood ingestion indicated that two mechanisms are involved: in addition to the alkalization promoted by CO2 volatilization there
is a minor contribution from a second mechanism not yet characterized. Some inferences concerning Leishmania development
and pH in the digestive tube are presented.
Key words: Lutzomyia longipalpis, midgut pH, pH control mechanisms, acidification, alkalization, Leishmania development.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2793pH of L. longipalpis midgut
contribute to the alkalization observed in mosquitoes and might be
involved in the pH change inside the midgut of phlebotomine sand
flies after a blood meal.
Considering the importance of pH to Leishmania development
and to digestive processes, the objectives of this study were to
measure the pH inside different parts of the L. longipalpis gut during
blood digestion in uninfected sand flies and to investigate some
midgut properties that could be involved in intestinal pH control in
unfed and blood-fed females. Finally, we offer a hypothesis
concerning the pH in the phlebotomine gut and how it correlates
with Leishmania development from blood intake to transmission.
MATERIALS AND METHODS
Insects
All experiments were performed with females from a population of
L. longipalpis originating from Teresina, state of Piauí, Brazil, and
maintained as a closed colony according to the methodology
proposed by Modi and Tesh (Modi and Tesh, 1983).
Microelectrode construction and calibration
Glass micropipettes were prepared with capillaries (1mm diameter)
that were pulled on an electropuller (PP-83; Narishige, Tokyo,
Japan). The micropipette tips were strengthened by heat in a
microforge to tip final diameters of 15–25μm. These micropipettes
were stored under desiccant conditions until microelectrode
construction. The construction of single-barrelled turgor-resistant
microelectrodes sensitive to H+ was carried out according to previous
studies (Gibbon and Kropf, 1993; Billker et al., 2000) with some
modifications. Ionophore–polyvinyl chloride (PVC) mixture was
obtained by mixing 3μl of H+ ionophore cocktail A (Fluka,
Ronkonkoma, NY, USA) with 7μl of a 0.075% solution of PVC
(Fluka, Buchs, Switzerland) dissolved in tetrahydrofuran (SigmaAldrich, Milwaukee, WI, USA). H+-sensitive microelectrodes were
prepared by backfilling the pulled micropipettes with about 1–2μl of
the ionophore–PVC mixture prepared just before use. The mixture
was introduced into micropipettes with plastic pipette tips pulled in
a flame in order to produce hollow and flexible tubes fine enough to
penetrate the back side of the micropipettes. In order to evaporate all
tetrahydrofuran, microelectrodes were kept for about 7days in a
desiccator under vacuum. According to our experience, the preparation
of such large-tip, turgor-resistant microelectrodes does not require a
previous silanization step. Just prior to use, these H+-sensitive
microelectrodes were filled with 0.1mol l–1 Mes/Tris-base buffer
pH4.3 also containing 0.1mol l–1 KCl. Reference microelectrodes
were filled at the tip side by suction with about 2μl of 0.2% warmed
agarose dissolved in 3mol l–1 KCl solution. The reference electrodes
were then backfilled with 3moll–1 KCl solution. Both the H+-sensitive
and the reference microelectrodes were connected through a Ag–AgCl
wire to a high impedance electrometer (pH meter 26; Radiometer,
Copenhagen, Denmark). Electrodes were calibrated at four points
between pH7.0 and 8.5 using 0.05mol l–1 standard buffer solutions
(potassium phosphate monobasic/NaOH pH 7.0 and 7.5; Trisbase/HCl pH8.0 and 8.5). Readings were considered only after
complete stabilization of the response, which usually occurred in
30–60s. Microelectrode measurements were rejected when calibration
curves before and after the insect impalement differed by more than
3mV (corresponding to 0.07pH units).
pH measurements in the abdominal midgut in blood-fed
females
Phlebotomine females were allowed to feed for about 20–30 min
on golden hamsters anaesthetized with Thiopentax® (CristáliaProdutos Farmacêuticos LTDA, SP, Brazil; 5% in saline;
0.1 ml 100 g–1). Fed insects were maintained at 25°C and 70%
relative humidity until use. Engorged females were individually
captured from 10 min to 32 h after feeding and briefly washed in
a 1% commercial detergent solution and subsequently in 0.9%
saline for a few seconds. Each washed female was rapidly
transferred to the surface of a 15% polyacrylamide gel embedded
with 0.9% saline, placed under a stereo microscope and
immediately impaled through the abdominal cuticle (Billker et al.,
2000) with both H+-sensitive and reference electrodes held by
micromanipulators. The readings were manually annotated and the
measured values (mV) were transformed into pH units using the
respective calibration curves.
pH measurements in the thoracic midgut in blood-fed females
pH of the thoracic midgut (TM) in blood-fed females (3–5 days
old) was measured with vital pH indicator dyes as already
described (Gontijo et al., 1998). Briefly, unbuffered solutions
(about 0.1%) of the indicator dyes Bromothymol Blue or
Bromocresol Purple were prepared individually by dissolving the
dyes in 10% sucrose solution. The Bromothymol Blue and
Bromocresol Purple solutions were adjusted to pH 7.0 and 6.5,
respectively, and immediately offered, soaked onto pieces of
cotton, to recently blood-fed females. Twenty hours later, the
females were dissected and the colours in the TM were compared
with standard solutions prepared with the same dyes at different
pH values covering 0.5 unit intervals.
α-Glucosidase assay and distribution along the midgut in
unfed females
Fifteen 3–4dayold fasted females were dissected in 0.9% saline.
Their digestive tracts were separated into TM and abdominal midgut
(AM) and were transferred to different microcentrifuge tubes
containing 250 or 100μl of 1% aqueous Triton X-100, respectively.
A 50μl sample of this material containing solubilized enzyme (a
volume containing 0.2 TM or 0.5 AM) was transferred to another
tube containing 200μl of 0.25mol l–1 Mes/NaOH buffer pH6.0. The
reaction was initiated by addition of 250μl of 12mmol l–1 pnitrophenyl α-D-glucopyranoside (Sigma, St Louis, MO, USA), a
synthetic substrate for α-glucosidases, dissolved in water (final
concentration 6mmol l–1). The tubes were incubated for 1h at 30°C
and the reactions were stopped by addition of 1ml of 0.375mol l–1
glycine/NaOH buffer pH 10.5. Readings were taken using a
spectrophotometer (Shimadzu UV-1650PC, Columbia, MD, USA)
at 400nm. Blanks were prepared with 50μl of 1% aqueous Triton
X-100 without any midgut material.
Effects of volatilization of CO2 on AM pH of blood-fed females
In order to investigate the contribution of CO2 loss to the
alkalization observed in blood-fed sand flies, 10ml of human blood
was collected in the presence of heparin and transferred to a 50 ml
Erlenmeyer flask, which was exposed for 1 h to atmospheric air
under continuous agitation (approximately 60cyclesmin–1). During
this period, the pH rose from 7.4 to 8.10±0.12 (N=17). After
alkalization, the pH was adjusted to 7.4 by careful addition of
1 mmol l–1 HCl dissolved in 0.9% NaCl solution and the blood
was offered to 3–4 day old fasted females (no sugar was offered
to these insects) in an artificial feeding apparatus at 37°C using
chick skins (Bastien, 1990). pH measurements were accomplished
in the midgut using H+-sensitive microelectrodes during the initial
phase (2–6 h) after ingestion as well as 24–28 h post-ingestion of
the ‘CO2-depleted’ blood.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2794
Forced feeding
Forced feeding was performed as described previously (Hertig and
Mcconnel, 1963; Anez et al., 1997). Microcapillaries were
prepared by constricting the extremities of glass capillary tubes,
in an alcohol flame, in order to permit the introduction of the insect
mouthparts (except the labium) into the narrowed channel obtained.
This procedure triggers a reflex that forces the insect to ingest the
liquid inside the capillary, probably as it occurs in natural bloodfeeding mode. A piece of modelling clay was used as a support
for the capillary tube, which could be moved as necessary in order
to be finely adjusted to the mouthparts of the insect. During the
forced-feeding procedure, each female (3–5 days old) was
maintained in an adequate position under a stereo microscope on
the tip of a plastic tube (3 mm diameter) covered with a piece of
fabric. The insect was kept immobilized by means of continuous
suction provided by a vacuum pump connected to the tube (Anez
et al., 1997).
pH measurements in the midgut of unfed females challenged
with buffered solutions and acetazolamide
The ability of the midgut to maintain its pH (pH6) in unfed females
was investigated by forced ingestion of strongly buffered solutions
containing pH indicator dyes. The indicator dyes Bromothymol Blue
and Bromocresol Purple were dissolved, to a final concentration of
0.1%, in 0.16mol l–1 Hepes/NaOH pH7.5 or 0.16mol l–1 Mes/NaOH
pH5.0, respectively. The technique of forced feeding was used to
force the females to ingest about 0.5–1μl of the solutions mentioned
above. Immediately after ingestion, females were dissected and the
colours inside the diverticulum and midgut compared with standard
buffered solutions containing each dye as explained above. The same
solutions containing 1 mmol l–1 acetazolamide, an inhibitor of
carbonic anhydrase (del Pilar Corena, 2005), were also introduced
by forced feeding into the midgut of unfed females to evaluate the
involvement of this enzyme in the mechanism of pH maintenance.
Carbonic anhydrase expression in the midgut of
L. longipalpis – RNA extraction, cDNA synthesis and PCR
To test for the presence of carbonic anhydrase in the midgut cells,
seventeen 4dayold females fed only with sucrose were dissected
and their midguts collected. Total RNA was extracted using an
RNeasy micro kit (Qiagen, Valencia, CA, USA) according to the
manufacturer’s instructions. The RNA was eluted in 16μl of MilliQ water and used for cDNA synthesis with 0.5μg of oligo-dT primer
(Promega, Madison, WI, USA) and the M-MLV reverse
transcriptase system (Promega) in a final volume of 25μl. The
nucleotide sequence of the carbonic anhydrase from A. aegypti
(GenBank accession no. AF395662) and the putative cytoplasmic
carbonic anhydrase from Anopheles gambiae (Giles 1902) (GenBank
accession no. DQ518576) were blasted against The Wellcome Trust
Sanger Institute L. longipalpis gene database (Hertz-Fowler et al.,
2004) in order to find sequences from L. longipalpis. Among the
blast results, two sequences with probability values higher than
1.0e–25 were chosen, NSFM-137e02.q1k and NSFM-126c10.q1k.
Each of these sequences was used to design primer pairs – forward:
5-gca ttt aac ggt ggt gct tt-3/reverse: 5-cat cct tat tgg cca ctg ct3; and forward: 5-tgc tgg aga att gca tct tg-3/reverse: 5-tgg tgg
acg gta gtt gtt ga-3. PCR was carried out for 35 cycles (94°C for
30s, 57°C for 30s, 72°C for 45s) with 1μl of the cDNA in addition
to 500nmol l–1 of each primer, 200μmol l–1 of each dNTP and 1U
of Taq Phoneutria® (Phoneutria, Belo Horizonte, MG, Brazil) in a
final volume of 20μl. The products were analysed by 2% agarose
gel electrophoresis.
V. C. Santos and others
Statistics
Fisher’s test was applied to the experiments in which the normal
midgut pH was challenged with buffered solutions to investigate
the effect of acetazolamide. For all other occasions, when pertinent,
Student’s t-test was applied. All data are presented as means ± s.d.
RESULTS
In normal conditions, L. longipalpis takes about 40–45h to complete
blood digestion. According to the data presented in Fig.1, in the
first 32h of digestion the pH slowly decreased from 8.15±0.31
(considering the data obtained in the first 10h post-blood ingestion)
to 7.70±0.17 (considering the data obtained at ≥24h post-blood
ingestion; P=0.03) in the AM of blood-fed females.
Despite the alkaline environment inside the AM during blood
digestion, the pH in the TM was between 5.5 and 6 at 20h after
feeding (Table1, Fig.2). The acidic pH inside the TM is consistent
with a site responsible for sucrose hydrolysis. According to Gontijo
and colleagues, the α-glucosidases of starved females are membranebound enzymes responsible for sucrose digestion in the midgut of
L. longipalpis with an optimum pH near 5.8 (Gontijo et al., 1998).
In agreement with this, the α-glucolytic activity measured in the
TM of unfed females was significantly higher
(1.806±0.532 OD h–1 TM–1) than that of the AM
(0.610±0301ODh–1 AM–1; P<0.001).
Table2 summarizes the results concerning the contribution of CO2
volatilization to the pH in the AM. The volatilization of CO2 from
10ml of blood in vitro was enough to alkalize the pH from 7.4 to
8.10±0.12 (N=17) after 1h of exposure to the air. Longer exposures
did not increase the alkalinity of the blood (data not shown). In
order to investigate the contribution of CO2 volatilization to the
alkalization observed after a blood meal, females were fed with
‘CO2-depleted’ blood, the pH of which was previously adjusted to
approximately 7.4 (7.39±0.03). In these females, the pH measured
at 2–6h post-blood ingestion was 7.34±0.48 (N=5). This pH value
was not significantly different from pH7.39 (P=0.75). At 24–28h
post-ingestion, the pH inside the AM was 7.56±0.27 (N=5),
significantly higher than pH7.39 (P=0.04).
To investigate the ‘buffering’ ability of the L. longipalpis midgut,
unfed females were challenged by forced ingestion of highly
buffered solutions containing pH indicator dyes. In these
experiments, females were challenged with 0.16mol l–1 Hepes
Time after bloodmeal (h)
0 10 15 20 25 30 35
pH u ni ts 6.0
6.5
7.0
7.5
8.0
8.5
9.0
5
Fig. 1. pH in the abdominal midgut during blood digestion in uninfected
females of L. longipalpis. Each point is from a separate experiment in
which pH was measured using H+-sensitive microeletrodes.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2795pH of L. longipalpis midgut
pH7.5 containing Bromothymol Blue or with 0.16mol l–1 Mes
pH5.0 containing Bromocresol Purple. The results are shown in
Tables 3 and 4, respectively. In both cases, the pH inside the TM
and AM showed an evident tendency to return to normal (pH6)
despite the presence of the buffers. The presence of acetazolamide,
a carbonic anhydrase inhibitor, was significantly effective in
diminishing the ability of the TM and AM to return to the normal
pH values when challenged with 0.16mol l–1 Hepes pH7.5. The
effect of acetazolamide in the females challenged with 0.16mol l–1
Mes pH5.0 was less pronounced in the AM but was statistically
significant in the TM. In contrast, the pH inside the diverticulum
was the same as that of the ingested solutions and was not affected
by acetazolamide.
In some insects, the intracellular enzyme carbonic anhydrase is
involved in CO2 hydration and consequently in the production of
H+ and HCO3– ions that could be used by the midgut cells for midgut
pH control (del Pilar Corena et al., 2004; del Pilar Corena et al.,
2005). To test for the presence of transcripts of carbonic anhydrase,
RT-PCR was performed using mRNA from the midgut of unfed L.
longipalpis females. The primers used for PCR were designed based
on two mRNA sequences of L. longipalpis with 68% homology to
an A. gambiae carbonic anhydrase and 70% homology to an A.
aegypti cytoplasmic carbonic anhydrase. The sequence alignment,
in addition to the RT-PCR results presented in Fig.3, suggest that
the L. longipalpis midgut could produce more than one isoform of
carbonic anhydrase-like transcript. However, these data should be
confirmed by further experiments.
DISCUSSION
In 2000, Billker and colleagues observed that one drop of blood
exposed to the air rapidly reaches pH8 as the CO2 volatilizes and
equilibrates to the atmospheric concentration of CO2 (Billker et al.,
2000). In their study, the CO2 volatilization was considered the sole
cause of the alkalization phenomenon in the midgut of two mosquito
species A. aegypti and A. stephensi, in which the pH in the AM
30min after blood ingestion was 7.52 and 7.58, respectively. In
contrast to these results, del Pilar Corena and colleagues observed
a remarkable alkalization in the AM of adult mosquitoes after
ingestion of a solution prepared with fetal calf serum and culture
medium containing pH indicator dyes (del Pilar Corena et al., 2005).
Although this method did not allow exact measurement of the pH,
it was clear that the AM was alkalized to the range 8–9.5 or 8.5–9.5
depending on the mosquito species. It is important to emphasize
that in this case the alkalization could not be attributed to the CO2
volatilization. The authors also observed that this alkalization
process was impaired by the use of acetazolamide and
methazolamide, which are carbonic anhydrase inhibitors (del Pilar
Corena et al., 2005). It should be noted that the specificity of this
kind of inhibitor is not absolute and other activities could also be
influenced.
In agreement with the results of del Pilar Corena and colleagues
(del Pilar Corena et al., 2005), we found that the pH in L. longipalpis
AM just after blood ingestion rapidly increased to values above 8
(Fig.1). On the other hand, this alkalization observed after a blood
meal could be attributed principally to the CO2 volatilization
mechanism. A minor contribution could be attributed to a second,
unknown mechanism as can be inferred from the data presented in
Table2. Taking the CO2 out of the blood (in vitro) caused an increase
in the pH to 8.10±0.12. Before the blood was offered to the females,
the pH was adjusted to approximately 7.4 and even without the CO2
to help the alkalization, the pH of the AM increased significantly
from 7.39±0.03 to 7.56±0.27, 24h after blood ingestion (P=0.04).
Evidently, the contribution of this second mechanism to the pH
change was lower when compared with that promoted by CO2
volatilization and was not observed in the first hours after blood
ingestion (Table2). This second mechanism seems similar to that
observed by del Pilar Corena and coworkers mentioned above.
Although the buffering system in the midgut of unfed females is
very efficient at maintaining pH6 (Tables2 and 3), the ingestion of
blood is able to switch off this mechanism. This effect seems to be
immediate, because in the first 10min after blood ingestion the pH
in the AM increased considerably, as shown in the first data points
of Fig.1. It is possible that free amino acids or even other molecules
from the plasma are responsible for shutting down the pH 6
maintenance system and for triggering the alkalization mechanism.
This physiological condition is probably maintained while amino
acids are absorbed by the intestinal epithelium as a consequence of
blood digestion by digestive proteases.
The pH5.5–6.0, measured in the TM during blood digestion
(Fig.2; Table1) indicates that the pH6 maintenance mechanism is
switched off in the midgut in a localized manner, i.e. only where
the blood is located, such as in the interior of the AM.
Whilst it is important to maintain an alkaline pH in the AM, at
the same time it is necessary to keep sucrose digestion working in
order to digest and use the ingested sugar meal. In L. longipalpis,
Table 1. pH in the thoracic midgut during blood digestion measured with pH indicator dyes
pH range 5.5 No. of TM 37 6 5 0 48
Unbuffered solutions (about 0.1%) of the indicator dyes Bromothymol Blue or Bromocresol Purple were prepared in 10% sucrose solution. The Bromothymol
Blue and Bromocresol Purple solutions were adjusted to pH 7.0 and 6.5, respectively, and immediately offered, soaked onto pieces of cotton, to recently
blood-fed females. Twenty hours later, the females were dissected and the colours in the thoracic midgut (TM) were compared with standard solutions
prepared with the same dyes at different pH values covering 0.5 unit intervals.
Fig. 2. Anatomy of L. longipalpis gut and pH in different parts of the midgut
during the first 10 h (a) and 24 h (b) after blood ingestion. TM, thoracic
midgut; AM, abdominal midgut; D, diverticulum filled with sugar solution; H,
hindgut.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2796
this problem was solved by maintenance of the pH at 5.5–6.0 in the
TM, even when the pH in the AM was alkaline. This pH range in
the TM is perfect for sucrose digestion by the L. longipalpis digestive
α-glucosidase, at the optimal pH of 5.8 (Gontijo et al., 1998). In
this manner, the simultaneous digestion of proteins and
carbohydrates can occur in distinct parts of the midgut with different
pH values. The data presented here show that most of the αglucolytic activity is in the TM.
In L. longipalpis, the α-glucosidase is more efficient in slightly
acidic conditions, even when the enzyme is obtained from insects
that are digesting blood (data not shown). In contrast, in Phlebotomus
langeroni, the α-glucolytic activity described in blood-fed females
has an optimal pH between 7 and 7.5 (Dillon and el-Kordy, 1997).
These data indicate important physiological differences between
these two phlebotomine species that deserve to be studied in more
detail.
Phlebotomus papatasi (and probably other phlebotomine species)
can sometimes ingest starch grains when they bite plants to obtain
sap (Schlein and Warburg, 1986). These grains are apparently
stocked in the diverticulum where the pH is not controlled by the
insect. Probably, the salivary α-amylase (Ribeiro et al., 2000), which
works well at pH 7 and is ingested with the saliva when the insects
V. C. Santos and others
ingest sugar (Cavalcante et al., 2006), digests the starch present in
the diverticulum, making this carbohydrate an alternative nutrient
for the insect.
The presence of transcripts of carbonic anhydrases in the midgut
cells and the results obtained with acetazolamide indicate that this
enzyme may be involved in the pH control mechanism in unfed L.
longipalpis females and is probably also involved in pH control in
blood-fed ones. The enzyme carbonic anhydrase functions by
Fig. 3. Carbonic anhydrase expression in the midgut of unfed L. longipalpis
females. Lanes: MW, relative molecular mass marker; 1, transcript with
homology to A. gambiae carbonic anhydrase (427 bp); 2, transcript with
homology to A. aegypti cytoplasmic carbonic anhydrase (455 bp); 3, 18S
subunit of rRNA (468 bp).
Table 2. pH measurements from CO2-depleted blood before ingestion and in the abdominal midgut of L. longipalpis during digestion
CO2-depleted blood1 CO2-depleted blood after pH correction2 CO2-depleted blood 2–6 h after ingestion3 CO2-depleted blood 24–28 h after ingestion4
8.1±0.12 (N=17)a 7.39±0.03 (N=11)b 7.34±0.48 (N=5)b 7.56±0.27 (N=5)c
1pH of the blood measured after CO2 volatilization by in vitro exposure to the air for 1 h; 2pH of the blood after CO2 volatilization and adjustment with addition of
1 mmol l–1 HCl; 3pH measured in the abdominal midgut (AM) of L. longipalpis in vivo 2–6 h after ingestion of the CO2-depleted blood with pH previously
adjusted to 7.39; 4pH measured in the AM of L. longipalpis in vivo 24–28 h after ingestion of the CO2-depleted blood with pH previously adjusted to 7.39.
Results marked with different superscript letters are significantly different (P<0.05).
Table 3. pH measurements in the midgut of unfed females of L. longipalpis challenged with 160mmol l–1 Hepes pH 7.5-buffered solution with
or without acetazolamide
TM AM Diverticulum
No ACZ ACZ No ACZ ACZ No ACZ ACZ
pH range
pH6.0 22 8 22 5 1 1
6.0
6.5
7.0
Total no. 37 28 35 26 26 18
Statistics P=0.0230 P=0.0008 P=1.000
Fisherʼs test was used to investigate differences provoked by treatment with acetazolamide between the observations presenting pH6.0 and a group formed
by all other observations (with pH>6.0). P<0.05 was considered significant.
TM, thoracic midgut; AM, abdominal midgut; ACZ, with acetazolamide; No ACZ, without acetazolamide.
Table 4. pH measurements in the midgut of unfed females of L. longipalpis challenged with 160mmol l–1 Mes pH 5.0-buffered solution with
or without acetazolamide
TM AM Diverticulum
No ACZ ACZ No ACZ ACZ No ACZ ACZ
pH range
5.5 0 8 1 4 13 13
5.5
6.0
6.5
Total no. 35 22 29 19 15 13
Statistics P=0.0002 P=0.0724 P=0.4841
Fisherʼs test was used to investigate differences provoked by treatment with acetazolamide between the observations presenting pH6.0 and a group formed
by all other observations (with pH>6.0). P<0.05 was considered significant.
TM, thoracic midgut; AM, abdominal midgut; ACZ, with acetazolamide; No ACZ, without acetazolamide.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2797pH of L. longipalpis midgut
hydrating CO2 molecules in order to produce H+ and HCO3– ions.
The subsequent destiny of them, determined by different ion pumps,
could be responsible for alkalization or acidification of the midgut,
depending on the physiological conditions. In fact, it has already
been demonstrated that carbonic anhydrase is involved in pH control
in the midgut of mosquitoes (adults and larvae) (Corena et al., 2002;
del Pilar Corena et al., 2004; del Pilar Corena et al., 2005).
As well as carbonic anhydrase, V-ATPases are also involved in
pH regulation in insects. These enzymes, which are important
proteins able to pump H+ using ATP as an energy source, have
already been described in L. longipalpis midgut cells (RamalhoOrtigão et al., 2007). It is very likely that they also participate in
the mechanism involved in pH control, probably acting differently
in unfed and blood-fed females.
Cuprophilic and goblet cells are specialized cells involved in pH
regulation in the midgut of Diptera (Muscomorpha) and Lepidoptera
larvae, respectively (Terra, 1988; Terra et al., 1988; Lepier et al.,
1994). However, morphological studies did not identify any of these
cells in the adult midguts of phlebotomine sand flies, which are
Diptera (Nematocera) (Billingsley, 1990; Leite and Evangelista,
2001).
In phlebotomine sand flies, Leishmania parasites live exclusively
in the lumen of the gut where amastigotes ingested during blood
feeding differentiate into flagellates that are morphologically and
biochemically distinct from amastigotes (Bates, 1994; Handman,
2000). These forms, generally known as promastigotes, present
morphological and physiological differences throughout their
development (Bates and Rogers, 2004) that culminate in the
appearance of metacyclic promastigotes able to infect mammalian
hosts (Sacks and Perkins, 1984; Sacks, 1989). Since Leishmania
development occurs exclusively within the midgut of the vectors,
many biochemical and physiological characteristics of this
environment may decisively influence the development of these
parasites and thus the success of transmission. Previously, some
factors have been described that are probably capable of influencing
Leishmania development towards metacyclic forms in culture
medium: exhaustion of nutrients (Giannini, 1974; Sacks and Perkins,
1985) as observed in cultures in the stationary phase (Sacks, 1989);
higher concentrations of CO2 in the culture medium when compared
with usual cultures (Méndez, 1999); absence or low levels of factors
able to inhibit metacyclogenesis such as haemoglobin (Schlein and
Jacobson, 1994), haemin (Charlab et al., 1995) and
tetrahydrobiopterin (Cunningham et al., 2001); contact with
phlebotomine saliva, which only acts in the absence of haemin
(Charlab et al., 1995); and, finally, acidification of the medium
(Bates and Tetley, 1993).
On the other hand, in apparent contradiction related to the
importance of an acidic environment to metacyclogenesis,
promastigotes cultivated in different culture media grow better in
neutral or slightly alkaline pH (M. N. Melo, personal
communication). Furthermore, some enzymes responsible for
protein digestion in the sand fly gut, especially proteases similar to
trypsin and chymotrypsin, function better in an alkaline pH
(Mahmood and Borovsky, 1993; Gontijo et al., 1998). To clarify
this apparent contradiction and explain the development of
Leishmania in infected sand flies, the following hypothesis was
proposed (Gontijo et al., 1998): after an infective meal, an alkaline
pH and the availability of nutrients during protein digestion would
favour the multiplication of Leishmania. This in vivo phase of
development would correspond to the logarithmic growth phase in
culture medium. During this period, the alkaline pH and the
presence of haemoglobin and haemin would inhibit
metacyclogenesis, as haemoglobin (Schlein and Jacobson, 1994) and
haemin (Charlab et al., 1995) seem to inhibit differentiation when
present. When protein digestion is complete, haemoglobin and
haemin should no longer be present and the nutrients would be
practically exhausted, including tetrahydrobiopterin. At this moment,
the pH of the intestine should decrease, stimulating metacyclogenesis
as it occurs in vitro. In 1998, Gontijo and colleagues found the
midgut pH of unfed L. longipalpis females to be 6.0, proving that
the gut is acidic when blood is not being digested (Gontijo et al.,
1998). In addition to the acidification, the phlebotomine saliva could
act on Leishmania promastigotes in the absence of haemin, driving
them to a differentiation pathway, as proposed by Charlab and
colleagues (Charlab et al., 1995). In fact, saliva is regularly ingested
during the sugar meal phase after blood digestion (Cavalcante et
al., 2006). This post-blood meal stage, characterized by low levels
of nutrients in an acidic environment, should correspond (in infected
phlebotomines) to the stationary growth phase in the culture
medium.
The alkaline pH measured in the L. longipalpis AM after blood
ingestion (Fig. 1; Table 2) is entirely in accordance with the
hypothesis proposed by Gontijo and colleagues described above
(Gontijo et al., 1998). Indeed, an alkaline environment is favourable
for the multiplication of Leishmania promastigote forms in culture,
where the parasite presents optimum growth in the pH range 7–8
(M. N. Melo, personal communication). Depending on the
Leishmania species, good growth rates, from 46% to 85% of the
growth rate obtained at the optimum pH, can be reached even at
pH8.5 (M. N. Melo, personal communication). After 24h, the AM
pH in uninfected females remains at around 7.7 (Fig.1), a pH
favourable to the growth of Leishmania once it is within the pH
range for optimal growth.
At present, there is no information about the pH in the midgut
of Leishmania-infected insects. However, it is possible that, in
females with a regular diet of carbohydrate, the pH is slightly lower
than in uninfected insects due to the carbohydrate metabolism from
promastigote forms. In fact, the phenomenon, called aerobic
fermentation, could generate acid catabolites from an incomplete
metabolism of monosaccharides such as glucose or fructose (Cazzulo
et al., 1985; Darling et al., 1987).
Further studies are being developed on this subject and we hope
soon to be able to propose a more complete model of how the
intestinal pH is controlled by phlebotomines and also to obtain
information about the pH variation in the midgut of L. longipalpis
females infected with L. infantum. The findings could provide a
better understanding of the development of this parasite in its vector.
We are thankful to Dr Maria Norma Melo for the unpublished data concerning the
efficiency of Leishmania growth at different pH in vitro. This work was supported
by Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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