Carbonic anhydrase in the adult mosquito midgut
1 The Whitney Laboratory, University of Florida, Saint Augustine, FL 32080,
USA
2 Arthropod-Borne and Infectious Diseases Laboratory, Department of
Microbiology, Immunology and Pathology, Colorado State University, Fort
Collins, CO 80523, USA
3 Department of Biochemistry, University of North Florida, Jacksonville, FL
32224, USA
4 Department of Biochemistry Cell and Molecular Biology, Drake University,
Des Moines, IA 50311, USA
5 Department of Pharmacology and Therapeutics, University of Florida,
Gainesville, FL 32611, USA
* Author for correspondence (e-mail: pjl{at}whitney.ufl.edu)
Accepted 7 June 2005
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Summary |
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Key words: mosquito, carbonic anhydrase, histochemistry, midgut pH, carbonic anhydrase inhibitor
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Introduction |
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Although the pH inside the larval midgut has been described in detail
(Boudko et al., 2001;
Charles and de Barjac, 1981
;
Dadd, 1975
;
Ramsay, 1950
), a comprehensive
study on the pH inside the adult mosquito midgut has not been presented. The
importance of maintaining pH for physiological processes that occur inside the
larval midgut and in survival of the larvae has been described recently
(Boudko et al., 2001
;
Corena et al., 2002
).
Little is known about pH maintenance inside the adult mosquito midgut.
Although the presence of trypsin and aminopeptidase, as well as esterases and
chitinase, has been demonstrated in the adult mosquito midgut
(Lemos et al., 1996;
Mourya et al., 2003
;
Okuda et al., 2002
;
Villalon et al., 2003
), there
are no reports on the presence of CA and the role of this enzyme in the
maintenance of pH inside the midgut. We have previously localized, cloned and
measured CA content in the midgut of larval Ae. aegypti
(Corena et al., 2002
) as well
as in other species of mosquito larvae
(Corena et al., 2004
). We have
found differences in CA distribution among different species of larvae.
However, mosquito larvae are morphologically and physiologically different
from adult mosquitoes. The purpose of this study was to determine the pH,
localize and measure CA content in the anterior and posterior midgut of adult
male and female mosquitoes, and to determine if CA plays a role in the
maintenance of this pH. Additionally, this study aimed to determine if CA
distribution between male and female midguts, as well as between the anterior
and posterior regions of the midgut, was different. We used Hansson's
histochemical method (Hansson,
1967
) and the 18O-isotope exchange method coupled to
mass spectrometry (Silverman and Tu,
1986
) for this purpose. We have used these techniques to measure
CA activity in larval mosquitoes and found significant differences between the
anterior and posterior regions in several species of larvae
(Corena et al., 2004
).
Here we present for the first time a detailed study of pH inside the adult mosquito midgut and the localization and measurement of CA activity in the anterior and posterior regions of female and male midguts from seven different species (Ae. aegypti, Ae. albopictus, An. albimanus, An. quadrimaculatus, Cx. nigripalpus, Cx. quinquefasciatus and Oc. taeniorhynchus). We also present data on the importance of CA in the maintenance of pH inside the midgut.
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Materials and methods |
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Larvae were raised from eggs obtained from colonies maintained at the United States Department of Agriculture (USDA) in Gainesville, Florida. Larvae were reared in tapwater or 50% seawater (in the case of Oc. taeniorhynchus) at 25±1°C. Adults were allowed to emerge and were separated before mating occurred. Both males and females were maintained at 25±1°C and fed with 10% sucrose in water.
Solutions and test formulations
Hemolymph substitute solution (HSS) was prepared according to Clark et al.
(1999). The solution consisted
of 42.5 mmol l1 NaCl, 3.0 mmol l1 KCl, 0.6
mmol l1 MgSO4, 5.0 mmol CaCl2, 5.0
mmol l1 NaHCO3, 5.0 mmol l1
L-succinic acid, 5.0 mmol l1 L-malic
acid, 5.0 mmol l1 L-proline, 9.1 mmol
l1 L-glutamine, 8.7 mmol l1
L-histidine, 3.3 mmol l1 L-arginine,
10.0 mmol l1 dextrose, and 25 mmol l1
Hepes, adjusted to pH 7.0 with NaOH.
Artificial seawater (100%) was prepared freshly every time. Final concentrations were 411.04 mmol l1 NaCl, 9.94 mmol l1 KCl, 10.25 mmol l1 CaCl2, 53.6 mmol l1 MgCl2, 28.24 mmol l1 Na2SO4; the pH was adjusted to 8.18.3 with NaOH. This stock solution was diluted to prepare 50% artificial seawater, used as a culture medium.
CA inhibitors [methazolamide (MTZ) and acetazolamide (ACZ)], dimethylsulfoxide (DMSO), adenosine triphosphate (ATP), sodium bicarbonate, Cresol Red, Phenol Red, Neutral Red, Thymol Blue and Dulbecco's phosphate buffered saline were obtained from Sigma-Aldrich Corp. (St Louis, MO, USA). Solutions of the inhibitors were prepared in DMSO to final concentrations of either 102 mol l1 or 103 mol l1 and used as stock solutions to prepare those of lower concentration by diluting aliquot samples of these stocks in distilled water. Protease inhibitor cocktail was obtained from Sigma-Aldrich Corp. The cocktail contained 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64, bestatin, leupeptin and aprotinin, with no metal chelators. Fetal bovine serum (FBS) was obtained from Gemini (Biotech, Alachua, FL, USA), minimum essential medium (MEM) from Gibco (Grand Island, NY, USA) and non-essential amino acids (Cellgro) from Mediatech, Inc. (Herndon, VA, USA).
pH measurements using indicators
Female mosquitoes were fed meals containing pH indicator in `media mixture'
(see below) for pH measurements inside the midgut. In some cases meals
contained 104 mol l1 of MTZ or/and ACZ for
pH measurements in the presence of CA inhibitors. The mosquitoes were allowed
to feed on the mixture at 37°C using a Parafilm® membrane on a glass
feeder. After 30 min, based on an assumed normal feeding time of
2.519.5 min (Reisen and Emory,
1976), mosquitoes were anesthetized at 4°C for 10 min, placed
on ice, dissected and photographed at room temperature. Each meal was prepared
using 20% FBS, 2x MEM, 0.02% pH indicator prepared in Dulbecco's
phosphate buffer saline, 1 mmol l1 ATP as phagostimulant,
1x non-essential amino acids, and 0.25% sodium bicarbonate, at a final
pH of 7.27.4, as `media mixture'. DMSO control included media mixture,
0.02% pH indicator and 0.001% DMSO. Acid control was prepared using SfII900
(pH=5.6) media instead of MEM. Finally, a positive control included a mixture
of 104 mol l1 MTZ and ACZ prepared in
DMSO.
Midguts of mosquitoes fed on meals containing Cresol Red and CA inhibitors (or no inhibitor) were dissected and observed under a stereoscopic microscope. Images were obtained using the Picture Frame v 1.01 software set in the monochromatic mode also. Images were saved at TIF-8 bit format to be analyzed using the toolbox mode of the ImageQuant TL software from Amersham-Biosciences®. The grey TIF-8 bit pictures from the midguts were used for image analysis. Graph and statistical analysis were made using GraphPad® software from Prism®.
CA histochemistry
Histochemistry was performed by the same method used to localize CA
activity in mosquito larvae (Corena et
al., 2002). Adult mosquitoes (males and females separately) were
cold anesthetized and dissected. 1525 whole midguts from males and
females from each species were dissected in HSS. The midguts were incubated
overnight at 4°C in 3% gluteraldehyde in 0.1 mol l1
sodium phosphate buffer, pH 7.3. The following day, the midguts were rinsed
three times with 0.1 mol l1 sodium phosphate buffer (pH 7.3)
followed by a 5 minincubation in a solution made by combining 17 ml of
solution A (1 ml 0.1 mol l1 solution of CoSO4
mixed with 6 ml 0.5 mol l1 H2SO4 and
10 ml 0.066 mol l1 KH2PO4) with 40 ml
of solution B (0.75 g NaHCO3 in 40 ml distilled water). After
incubation, the guts were rinsed again in sodium phosphate buffer and
incubated in 0.5% (NH4)2S for 2 min followed by a rinse
with distilled water. The midguts were placed on depression slides and
digitally imaged using a Zeiss Axiovert 135 TV inverted microscope (Carl Zeiss
Inc., Thernwood, NY, USA) equipped with a CCD camera.
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Results |
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Only two pH indicators indicated a change in pH due to the presence of CA inhibitors, Cresol Red and Thymol Blue (Fig. 3). The most noticeable differences were observed with Cresol Red and a mixture of the two CA inhibitors, MTZ and ACZ (Fig. 4). The effect of MTZ was quantitated using color intensity comparisons of 10 treated vs untreated midguts paired in TIF-8 bit grey scale format from two independent experiments (Fig. 5A). Significant differences in color intensity were observed between the methazolamide-treated midguts and the non-treated ones (Fig. 5B).
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Histochemistry of female midguts
Using Hansson's histochemical method, a black precipitate that appears as
darkening of the tissue is produced at the sites of CA enzymatic activity.
Since this technique shows activity directly in the tissue, the results
include both cytosolic and membrane-bound CA activity. Dissected midguts
representative of females from each species are shown in
Fig. 6. We observed differences
among the different species used in this study. Comparison of female midguts
from Cx. nigripalpus, An. quadrimaculatus, Ae. albopictus and Cx.
quinquefasciatus showed that CA is preferentially localized in the
posterior midgut (Fig. 6A,B,C and
D, respectively) as indicated by darkening of the tissue.
Histochemistry of Ae. aegypti and Oc. taeniorhynchus female
midguts revealed uniform staining throughout the midgut in all the specimens
dissected (Fig. 6E,F). Female
Ae. aegypti midguts appeared darker than the midguts from other
species.
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Total protein concentration
Total soluble protein for anterior and posterior midgut homogenates was
calculated for each species after homogenization and centrifugation of the
tissue. The values obtained were corrected for the amount of protease
inhibitors added to each individual sample. Although we did not calculate the
amount of protein per midgut, the majority of the species tested exhibited the
highest soluble protein content in the posterior midgut (results not shown).
Since different species vary in size, a comparison of the protein content from
one portion of the gut in one species to the same portion in another species
is imprecise.
The highest amount of total soluble protein was found in posterior midgut homogenates of An. quadrimaculatus females. The second highest amount was found in anterior midgut homogenates of females of the same species. In the males, the highest total soluble protein content was found in the anterior midgut of An. quadrimaculatus followed by the anterior midgut of Cx. nigripalpus.
18O exchange method to measure CA activity
Since Hansson's histochemical method provides a qualitative estimate in
terms of the total amount of CA activity (both soluble and membrane bound)
present in each region of the midgut, a quantitative technique was necessary
in order to determine the amount of activity present in each tissue. The
18O isotope exchange method has been used previously to determine
CA activity in larval midguts (Corena et
al., 2004). Although very sensitive, this method is used only to
quantify soluble CA, and discrepancies between Hansson's qualitative method
and the 18O isotope exchange technique are expected. These
differences are probably the result of the loss of membrane-bound CA in the
pellet after centrifugation. Therefore, the amounts of CA activity calculated
with the 18O isotope exchange technique in this manuscript refer
only to cytosolic CA. We have observed a correlation between Hansson's
histochemical method and the 18O isotope exchange method in the
larvae but this might not hold true for the adult mosquito, as adults (flying
insects) and larvae (aquatic insects) are very different.
In terms of our findings using the 18O isotope exchange method, CA activity in the adult mosquito midgut seems to be preferentially associated with the posterior midgut in the females of most of the species tested. This also seems to be true for the males of most species. We were able to detect CA activity in the anterior midgut of females in only three species: Ae. aegypti (0.6%), Ae. albopictus (0.02%) and Cx. nigripalpus (0.08%) (Fig. 8A). We were unable to detect CA activity in the posterior midgut of An. albimanus and Cx. nigripalpus females (Fig. 8B). We were unable to detect CA activity in the anterior midguts of the males of the species tested in this study. We were unable to detect CA in the posterior midgut of An. quadrimaculatus and Ae. aegypti males (Fig. 8C).
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Discussion |
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Mosquito blood meal pH has been determined in situ using
ion-selective microelectrode measurements. Using this technique, blood meal pH
has been shown to increase from 7.40 to 7.52 in Ae. aegypti females
and to 7.58 in An. stephensi
(Billker et al., 2000).
Measurements of pH inside the mosquito midgut have been documented in the
larvae and the data collected has relied on the use of pH indicators, using
particles to assist ingestion (Zhuang et
al., 1999
). Inside the anterior larval midgut, pH values have been
reported to be in the range 9.511
(Clements, 1992
). Maintenance
of this high pH in the anterior larval midgut has subsequently been associated
with a high concentration of bicarbonate/carbonate ions and hence presence of
CA (Boudko et al., 2001b; Corena et al.,
2002
,
2004
).
Is the pH inside the adult mosquito midgut similar to that of the larvae where alkalization is higher in the anterior region and decreases in the posterior? To answer this question, we determined the pH inside the midgut of female Ae. aegypti, An. gambiae and Cx. tarsalis adult mosquitoes.
Our results using pH indicators suggest that the pH inside the adult
posterior midgut in female Ae. aegypti is between 8.5 and 9.5. In the
other two species it was in the range 8.09.5. It is not surprising to
find differences in pH inside the midgut of mosquitoes from different species.
We have observed such differences in the midgut of larvae from several species
and these observations have also been reported by other investigators
(Clements, 1992). However, when
comparing these results with those previously obtained in the larval midgut,
we can conclude that the pH inside the anterior adult female mosquito midgut
does not reach the high values found in the anterior midgut in the larvae
(pH>10). In fact, the adult female mosquito midgut presents the complete
opposite: a more alkaline posterior midgut and a more acidic anterior midgut.
We have focused our observations in the posterior midgut since this is the
site of storage of the blood meal.
Does CA play a role in the maintenance of this pH as it has been suggested in the larvae? We have used CA-specific inhibitors to determine if the pH is affected by the absence of CA activity. We must consider that mosquitoes were artificially fed on pH indicator and CA inhibitor at the same time; thus the change on the overall midgut pH reflects only the inhibitory effect that takes place a short time after the meal is ingested. A background effect of CA activity at the time of the meal is to be expected. Treatment with CA inhibitors resulted in a decrease in pH inside the midgut from the initial values (8.59.5) to 7.5 and 8.0. Analysis of differences in color intensity of 8-bit grey scale images revealed significant differences (P>0.0001) between the treated and the untreated midguts. We can conclude that CA is important in maintaining the pH inside the adult female mosquito midgut. Little is known about the mechanism of pH maintenance inside the adult mosquito midgut but our observations suggest that CA is crucial in the maintenance of pH within the midgut in all the species tested.
How does MTZ affect alkalization of the midgut? It is possible that this compound inhibits the production of bicarbonate by inhibiting one (or more CA isozymes), therefore interfering with the ion transport processes that occur in the midgut epithelium, ultimately altering the alkalization mechanism and leading to changes in pH.
Is the localization of CA related to the mechanisms of ion regulation in
the midgut for each particular species? A detailed analysis of CA isozyme
distribution in the midgut for each species and its relationship with ion
transport mechanisms will be necessary in order to answer this question. We
observed a significant decrease in pH on using a combination of ACZ and MTZ.
Since these two compounds inhibit different CA isozymes to various degrees, it
is possible that more than one isozyme is present in the adult mosquito
midgut. At least 14 CA isozymes have been identified in the An.
gambiae genome (Holt et al.,
2002). It is possible that multiple CA isozymes are present in the
adult mosquito midgut and contributing to the mechanisms of ion transport.
However, further studies are necessary to determine the localization and
characterization of these isozymes. A correlation between CA localization and
ion transport in the midgut could be made afterwards by using self-referencing
ion selective (SERIS) microelectrodes to measure ion fluxes in the midgut, as
has been demonstrated in the larvae (Boudko
et al., 2001
).
Is CA present in the midgut of adult mosquitoes? Are there any differences between species? CA localization by Hansson's histochemical method revealed that the enzyme is present in the midgut of all the species tested, with the exception of Oc. taeniorhynchus males. However, quantitative analysis using the 18O isotope exchange method revealed a small amount of CA activity in Oc. taeniorhynchus males. We can conclude that all the species tested exhibited CA activity in the midgut to various degrees.
We have previously observed a correlation between Hansson's histochemical
method and the 18O isotope exchange method in the localization of
CA in larval Ae. aegypti, Cx. quinquefasciatus, Cx. nigripalpus, Oc.
taeniorhynchus, Ae. albopictus and An. quadrimaculatus midgut
(Corena et al., 2002,
2004
). It is important to
clarify that there are at least 14 possible genes that code for CAs in the
An. gambiae genome and that in this study we only determined total
soluble protein in each of the midgut regions. Therefore, the values presented
for CA activity account only for cytosolic CA and not membrane-bound CA that
could have been lost in the pellet after centrifugation and prior to analysis.
To date, there is no direct evidence for the presence of membrane-bound CA in
the adult mosquito midgut, although it has been detected in Ae.
aegypti and An. gambiae larval midguts
(Seron et al., 2004
).
In terms of the CA activity in different species, we observed differences among the mosquito species tested. The highest enzymatic activity quantitated appeared to be associated with the posterior midgut in Cx. quinquefasciatus males, followed by the posterior and anterior midgut in Ae. aegypti females, respectively. We are able to conclude that CA distribution in the midgut of all the species of adult mosquitoes tested in this study appears to be species dependent.
Are there any differences between male and female adult mosquito midguts? The answer to this question is yes, not only are there differences between male and female mosquito midguts, but the differences are striking. We have observed differences not only in localization but also in quantitation in male and female midguts. CA localization was consistent in the posterior midgut in females from all species. The amount of CA activity measured using the 18O-isotope exchange method showed that CA is expressed in this tissue at different levels in different species. We were able to detect CA in the anterior midgut only in females from Ae. aegypti, Ae. albopictus and Cx. nigripalpus. We observed interesting results for males of all the species tested. Although we were able to measure total protein in all of the species, we were unable to detect CA activity in the anterior midgut of the males from the species tested. In contrast, we detected CA activity in the posterior midgut of males from most species with the exception of An. quadrimaculatus males.
In terms of CA localization in the anterior and posterior regions of the
adult midgut, it is apparent now that all the species tested exhibit CA
activity in the posterior midgut, although to various degrees. Since the size
of the midgut varies from species to species, we determined the total amount
of soluble protein present in the midguts to determine if our data were
influenced by the amount of total protein. Analysis of our results
demonstrated that there is no apparent relationship between the amount of
total protein and the CA activity in the midgut. For example, in An.
quadrimaculatus females, the total soluble protein content was 164.8
µg in 100 µl of tissue homogenate while the amount of CA activity
present in this homogenate was only 0.2% of the total protein. In contrast,
the lowest amount of soluble protein found for a female mosquito was that of
the anterior midgut of Ae. aegypti. However, the amount of CA
activity reached 1% of the total protein present in that homogenate. Therefore
our results indicate that there is no apparent relationship between the amount
of total protein and the amount of CA present in a particular region of the
midgut. We have previously made this observation in larvae from different
species of mosquitoes (Corena et al.,
2004).
As a result of our experiments, we can conclude that the pH inside the adult female mosquito midgut is between 8.0 and 9.5. Furthermore, CA content is not only species dependent but also dependent on the sex of the insect and the portion of the midgut being studied. Inhibition of CA in the adult female mosquito midgut resulted in a pH imbalance with possible inhibition of ion transport processes, which led to a decrease in the alkalinity of the midgut.
The relevance of these studies lies in the importance of maintaining this
pH in homeostasis and ion transport mechanisms, which are involved in the
rapid digestion of the blood meal, and their role in the malaria infection
process. Blood pH is regulated by the equilibrium between dissolved
CO2 and bicarbonate ions. The loss of CO2 that occurs
when infected blood is exposed to ambient conditions has long been known to
result in a pH increase large enough to induce malarial gametogenesis in
vitro (Chorine, 1933;
Bishop and Mc Connachie, 1956
;
Nijhout and Carter, 1978
). We
believe that the increase in pH observed in the mosquito midgut during
digestion of an infected blood meal is the result of a high bicarbonate
concentration generated by CA activity. We postulate that this CA activity
leads to the conversion of CO2 into bicarbonate, which in turn
contributes to the induction of gametogenesis of Plasmodium parasites
inside the midgut. We have begun to study the role of CA activity in the
development of Plasmodium parasites inside the mosquito midgut and
the effect of CA inhibition in the infection mechanism. Our results will be
published in the near future.
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Acknowledgments |
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References |
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---|
Billker, O., Miller, A. J. and Sinden, R. E. (2000). Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis. Parasitology 120,547 -551.[CrossRef][Medline]
Bishop, A. and McConnachie, E. W. (1956). A study of the factors affecting the emergence of the gametocytes of Plasmodium gallinaceum from the erythrocytes and the exflagellation of the male gametocytes. Parasitology 46,192 -215.[Medline]
Boudko, D. Y., Moroz, L. L., Harvey, W. R. and Linser, P. J.
(2001). Alkalinization by chloride/bicarbonate pathway in larval
mosquito midgut. Proc. Natl. Acad. Sci. USA
98,15354
-15359.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Charles, J. F. and de Barjac, H. (1981). [pH variations in the midgut of Aedes aegypti in relation to Bacillus thuringiensis var. israelensis (serotype H14) crystal intoxication]. Bull. Soc. Pathol. Exot. Filiales 74, 91-95.[Medline]
Chorine, V. (1933). Conditions qui régissent la fécondation de Plasmodium praecox. Arch. Institut Pasteur d'Algérie 11, 1-8.
Christophers, S. R. (1960). Aedes aegypti (L.), the Yellow Fever Mosquito; Its Life History, Bionomics and Structure. Cambridge: Cambridge University Press.
Clark, T. M., Koch, A. and Moffett, D. F.
(1999). The anterior and posterior `stomach' regions of larval
Aedes aegypti midgut: regional specialization of ion transport and
stimulation by 5-hydroxytryptamine. J. Exp. Biol.
202,247
-252.
Clements, A. N. (1992). The Biology of Mosquitoes. London, New York: Chapman and Hall.
Corena, M. del P., Seron, T. J., Lehman, H. K., Ochrietor, J.
D., Kohn, A., Tu, C. and Linser, P. J. (2002).
Carbonic anhydrase in the midgut of larval Aedes aegypti: cloning,
localization and inhibition. J. Exp. Biol.
205,591
-602.
Corena, M. D. P., Fiedler, M. M., VanEkeris, L., Tu, C. K., Silverman, D. N. and Linser, P. J. (2004). Alkalization of larval mosquito midgut and the role of carbonic anhydrase in different species of mosquitoes. Comp. Biochem. Physiol. 137C,207 -298.
Dadd, R. H. (1975). Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes. J. Insect Physiol. 21,1847 -1853.[CrossRef][Medline]
Galun, R., Friend, W. G. and Nudelman, S. (1988). Purinergic reception by culicine mosquitoes. J. Comp. Physiol. A 163,665 -670.[Medline]
Hansson, H. P. (1967). Histochemical demonstration of carbonic anhydrase activity. Histochemie 11,112 -128.[CrossRef][Medline]
Holt, R. A., Subramanian, G. M., Halpern, A., Sutton, G. G.,
Charlab, R., Nusskern, D. R., Wincker, P., Clark, A. G., Ribeiro, J.
M., Wides, R. et al. (2002). The genome sequence of the
malaria mosquito Anopheles gambiae. Science
298,129
-149.
Lemos, F. J., Cornel, A. J. and Jacobs-Lorena, M. (1996). Trypsin and aminopeptidase gene expression is affected by age and food composition in Anopheles gambiae. Insect Biochem. Mol. Biol. 26,651 -658.[CrossRef][Medline]
Mourya, D. T., Rohankhedkar, M. S., Yadav, P., Dighe, V. and Deobagkar, D. N. (2003). Enhanced esterase activity in salivary gland and midgut of Aedes aegypti mosquito infected with dengue-2 virus. Ind. J. Exp. Biol. 41, 91-93.[Medline]
Nijhout, M. M. and Carter, R. (1978). Gamete development in malaria parasites: bicarbonate-dependent stimulation by pH in vitro. Parasitology 76,39 -53.[Medline]
Nuttal, G. H. F. and Shipley, A. E. (1903). Studies in relation to malaria. II. The structure and biology of Anopheles (Anopheles maculipenis). J. Hyg. 3, 166-215.
Okuda, K., de Souza Caroci, A., Ribolla, P. E., de Bianchi, A. G. and Bijovsky, A. T. (2002). Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion. Tissue Cell 34,210 -219.[CrossRef][Medline]
Ramsay, J. A. (1950). Osmotic regulation in mosquito larvae. J. Exp. Biol. 27,145 -157.[Medline]
Reisen, W.K. and Emory, R. W. (1976). Blood feeding of Anopheles stephensi. Ann. Entomol. Soc. Am. 69,293 -298.
Seron, T. J., Hill, J. and Linser, P. J.
(2004). A GPI-linked carbonic anhydrase expressed in the larval
mosquito midgut. J. Exp. Biol.
207,4559
-4572.
Silverman, D. N. and Tu, C. K. (1986). Molecular basis of the oxygen exchange from CO2 catalyzed by carbonic anhydrase III from bovine skeletal muscle. Biochemistry 25,8402 -8408.[CrossRef][Medline]
Thompson, M. T. (1905). Alimentary canal of the mosquito. Proc. Boston Soc. Nat. Hist. 32,145 -202.
Villalon, J. M., Ghosh, A. and Jacobs-Lorena, M. (2003). The peritrophic matrix limits the rate of digestion in adult Anopheles stephensi and Aedes aegypti mosquitoes. J. Insect Physiol. 49,891 -895.[CrossRef][Medline]
Zhuang, Z., Linser, P. J. and Harvey, W. R.
(1999). Antibody to H(+) V-ATPase subunit E colocalizes with
portasomes in alkaline larval midgut of a freshwater mosquito (Aedes
aegypti). J. Exp. Biol. 202,2449
-2460.