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First published online November 17, 2006
Journal of Experimental Biology 209, 4638-4651 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02551
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P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti
Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521-0146, USA
* Author for correspondence at present address: Department of Biology, University of San Diego, 5998 Alcalá Park, San Diego, CA 92111, USA (e-mail: mpatrick{at}sandiego.edu)
Accepted 13 September 2006
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Summary |
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Key words: mosquito, larva, adult, Aedes aegypti, P-type Na+/K+-ATPase, V-type H+-ATPase, ion transport, osmoregulation
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Introduction |
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).
To deal with these insults to hemolymph homeostasis, larval and adult
mosquitoes are equipped to rapidly respond and restore water and ion balance.
Freshwater larvae eliminate excess water load by producing copious, dilute
urine through the coordinated activity of the Malpighian tubules and hindgut,
whereas the four anal papillae surrounding the anal opening are the primary
sites of Na+ and Cl- absorption. Through these
mechanisms of ion and water regulation hemolymph composition remains stable
(Bradley, 1994). When adults
ingest nectar, elimination of the water load is initiated within seconds of
feeding and occurs without a significant loss of ions. This process, as in the
larva, involves the Malpighian tubules and hindgut and results in rapid urine
production and excretion. When a female takes a blood meal, excess salts
(Na+, K+ and Cl-), which are absorbed across
the stomach, are rapidly eliminated by Malpighian tubules/hindgut activity
(Clements, 2000).
In order for the larva and adult to perform the above described
osmoregulatory functions, ions must be transported against their
electrochemical gradients. Primary urine production by the Malpighian tubules
of larval A. aegypti is the consequence of active K+ and
Na+ secretion (Cl- movement is considered to occur
passively) from the hemolymph into the tubule lumen, with water following down
its osmotic gradient (Clements,
2000). Prior to being excreted, ions are actively absorbed from
the urine by the rectal epithelium back into the hemolymph
(Ramsay, 1950). In the adult
female, excess Na+, K+, Cl- and water from a
blood meal are secreted from the hemolymph into the Malpighian tubules, the
active process being the movement of cations against their electrochemical
potentials (Beyenbach,
1995).
But what is the proximal source of energy driving this active transport in
the different osmoregulatory organs of mosquitoes? For many years, insect
physiologists examining ion transport have focused on the V-type
H+-ATPase and have consider it the major player in establishing
favourable, ion-motive gradients. This V-type H+-ATPase has been
characterized in Malpighian tubules of adult A. aegypti by
electrophysiological studies (Beyenbach et
al., 2000) and in larvae by immunlocalization assays
(Filippova et al., 1998). With
regards to other osmoregulatory organs, this pump has been localized
(Filippova et al., 1998;
Zhuang et al., 1999) and
characterized in vitro (Boudko et
el., 2001) as the generator of larval midgut alkalinity (pH 11)
whereas in the adult stomach, V-type H+-ATPase-overexpressing cells
correlate with the distribution of malaria parasite oocysts in both Aedes
aegypti and Anopheles gambiae
(Cociancich et al., 1999). By
contrast P-type Na+/K+-ATPase, long considered to be
driving ion transport in many osmoregulatory systems, such as the thick
ascending loop of vertebrate nephrons
(Greger and Kunzelmann, 1990)
and the gills of various aquatic invertebrates
(Lucu and Towle, 2003;
Onken et al., 2003), has not
garnered as much attention in mosquito ion transport systems despite the fact
that ouabain-sensitive P-type Na+/K+-ATPase activity has
been reported in adult A. aegypti Malpighian tubules
(Hegarty et al., 1991) and in
the midgut of Anopheles stephensi
(MacVicker et al., 1993). So
the question remains as to the relative importance of these two ATPases in
driving ion transport in each of the osmoregulatory tissues of larval and
adult A. aegypti.
This present study takes a first step in addressing this issue by surveying all of the tissues relevant to osmoregulation in A. aegypti larvae (gastric caeca, midgut, Malpighian tubules, rectum, anal papillae) and adults (stomach, Malpighian tubules, anterior intestine, rectum) in order to determine V-type H+-ATPase and P-type Na+/K+-ATPase distribution and expression patterns, at the protein and gene levels using immunohistochemistry and reverse-transcriptase PCR assays. Our findings, in conjunction with previous morphological and physiological studies, provides a foundation for understanding the relative importance of V-type H+-ATPase and P-type Na+/K+-ATPase in the disparate osmoregulatory challenges faced by A. aegypti at two stages of development.
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Materials and methods |
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Tissue collection for immunohistochemistry and RT-PCR
To obtain tissues for immunohistochemistry (whole-mount and paraffin
embedded) and RNA isolation for reverse transcriptase polymerase chain
reaction (RT-PCR) assay, fourth instar larvae were rinsed and dissected in
ice-cold phosphate-buffered saline (PBS). The entire larval gut (gastric
caeca, midgut, Malpighian tubules, hindgut, anal papillae) was removed and the
peritrophic membrane and contents thereof were removed. For whole-mount
immunohistochemistry, the entire gut was transferred to 4% paraformaldehyde
(PFA)/PBS solution and fixed overnight at 4°C. For paraffin embedding,
whole larvae were used with the heads removed and several incisions were made
in the cuticle along the body to ensure proper fixation in 4% PFA/PBS solution
at 4°C overnight. Tissues for RNA isolation, the midgut (including gastric
caeca), Malpighian tubules, hindgut, and the anal papillae were separated and
transferred into cryovials on dry ice. Tissues from 15-20 larvae were combined
in these tubes.
Adult female mosquitoes used in this experiment were 5-7 days post-eclosion. They were identified and separated from males after CO2 anesthesia. For whole-mount immunohistochemistry, the entire gut including the stomach, Malpighian tubules, anterior hindgut and rectum were dissected out in ice-cold PBS and fixed in 4% PFA/PBS solution overnight at 4°C. After rinsing in PBS, an incision along the longitudinal plane of the stomach was made. For tissues to be embedded in paraffin, the head, wings, legs and the last abdominal segment were removed and small tears in the abdominal cuticle were made to ensure penetration of the fixative. The thorax/abdomen was then fixed overnight in 4% PFA/PBS at 4°C. For RNA sampling, females were dissected in ice-cold PBS and the anterior midgut/stomach, Malpighian tubules, anterior intestine and the rectum were separated and transferred to cyrovials in dry ice. Tissues from 20-25 adult females were combined.
Immunohistochemistry of P-type Na+/K+-ATPase and V-type H+-ATPase in larval and adult female mosquitoes
To localize P-type Na+/K+-ATPase protein, the
monoclonal antibody, `a5', raised against the 
-subunit of avian P-type
Na+/K+-ATPase in mice by Dr Douglas Fambrough, was
employed. This antibody was obtained from the Developmental Studies Hybridoma
Bank (DSHB). This antibody was developed under the auspices of the National
Institute of Child Health and Human Development and maintained by The
University of Iowa, Department of Biological Sciences, Iowa City, IA 52242,
USA. To localize V-type H+-ATPase, a polyclonal serum antibody
raised against the B subunit of the V-type H+-ATPase of Culex
quinquefasciatus was employed
(Filippova et al., 1998).
For whole-mount immunohistochemistry, gut tissues were thoroughly rinsed in
PBS followed by a methanol dehydation/rehydration series. Tissues were rinsed
with PBS then blocked for 2 h at room temperature with PBS/0.1% Triton X-100
(PBT) including 2% bovine serum albumin (BSA). Tissues were incubated
overnight at 4°C in a 5 µg ml-1 solution of the primary
antibody a5 (avian Na+/K+-ATPase) and a 1:1000 dilution
of polyclonal serum antibody to the V-type H+-ATPase made with
PBT/1% BSA. An equivalent solution with supernatant NS-1 myeloma solution
(supplied by DSHB) and rabbit preimmune serum (1:1000 dilution) served as a
control for both antibodies. To remove unbound antibody, tissues were rinsed
several times with PBT/1% BSA/2% normal goat serum (NGS). Next, tissues were
incubated for 2 h at room temperature with Cy3-labeled goat anti-mouse
secondary antibody and Cy5-labeled goat anti-rabbit secondary antibody
(Jackson Immuno Research, CO, USA) at a 1:2000 dilution in PBT/1% BSA/2% NGS.
The Cy3 anti-mouse and Cy5 anti-rabbit secondary antibodies were developed to
have minimal cross reactivity with other species in the incubation medium.
Preliminary tests indicated that concurrent incubation of 5 and V-type
H+-ATPase antibodies and two secondary antibodies did not result in
cross reactivity. After further rinsing with PBT/1% BSA/2% NGS, the tissues
were mounted on slides using 90% glycerol/4% N-propyl gallate. The
slides were stored at -20°C in the dark.
The thorax/abdomen of larvae or adult female mosquitoes were rinsed thoroughly in PBS then put through an ethanol dehydration series. The tissues were incubated in 100% ethanol followed by 70% ethanol/30% xylene solution, then 30% ethanol/70% xylene for 1 h each and finally 100% xylene overnight at room temperature. The next day, finely chopped paraffin chips (Paraplast PlusTM) were added to the vials to approximately 50% of total volume. After 3-4 h, the xylene was changed and samples were incubated overnight at room temperature. The vials were then placed in an oven at 55-60°C. After the paraffin melted, half of the volume was replaced with melted paraffin and incubated at 55-60°C for 2-3 h. Next, the entire volume was replaced with fresh melted paraffin several times. The larval tissues and melted paraffin were then transferred to an embedding mold and allowed to solidify.
Sections (8 µm thick) of larval and adult tissue were made then mounted onto poly-lysine-coated slides and allowed to dry. Sections were dewaxed in 100% xylene, put through an ethanol rehydration series and finally washed with distilled water then transferred to PBT. Next the slides were blocked with PBT/2% BSA for 1-2 h at room temperature. Sections were incubated with the primary antibodies for V-type-ATPase and P-type Na+/K-ATPase (in PBT/1% BSA) at 4°C overnight. The next day, sections were washed with PBT/1% BSA/2% NGS and incubated with Cy3- and Cy5-labeled secondary antibodies for 2-3 h followed by rinsing with PBT/1% BSA/2% NGS. Finally the sections were mounted in glycerol/gelatin/Tris pH 7.4, with coverslips on top and stored at -20°C in the dark.
Whole mounts and tissue sections were examined by scanning confocal microscopy with a helium/neon laser (Zeiss LSM510 Axioplan 2; located in the Center of Advanced Microscopy and Microanalysis, University of California, Riverside). The tissues were analyzed with a 10x, 40x and 100x objectives. All images were imported into Adobe Photoshop for assembly and labelling.
Sequencing
cDNA libraries were made from isolated midgut and Malpighian tubules of
fourth instar larvae and adult female Aedes aegypti as previously
described (Jin et al., 2003).
Analysis of random clones from these cDNA libraries identified a number of
V-type H+-ATPase subunits, and P-type
Na+/K+-ATPase. These clones were fully sequenced in both
directions, and the nucleotide sequences deposited in GenBank.
RNA isolations and RT-PCR
Total RNA was extracted from the adult female stomach using TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) and further purified using RNeasy mini kit
clean up protocol (Qiagen, Valencia, CA, USA). For adult Malpighian tubules,
anterior hindgut, rectum and larval Malpighian tubules, hindgut, and anal
papillae, total RNA was isolated using the RNeasy mini kit animal tissue
protocol (Qiagen). RNA concentrations were determined (GeneQuant II
spectrophotometer, Pharmacia Biotech, Piscataway, NJ, USA).
cDNA was reverse transcribed from equal amounts of total RNA from the larval and adult tissues using Superscript II First Strand cDNA synthesis kit (Invitrogen) with oligo hexamers, and included no-RT controls for each tissue. Primers for P-type Na+/K+-ATPase subunit and V-type H+-ATPase B subunit gene expression were designed to lie in adjacent exons based on the cDNA sequences for A. aegypti and the Softberry exon prediction program (RNASPL; www.softberry.com) so as to minimize genomic DNA amplification. 18s rRNA gene expression was also determined for each tissue as a control. All PCR assays were designed with an annealing temperature of 55°C. PCR products were run on a 1.5% agarose/ethidium bromide gel and documented using a Fotodyne system (Hartland, WI, USA).
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The distribution of the ATPases in the Malpighian tubules was dependent on cell type and region (Fig. 2). The V-type H+-ATPase was expressed only in the principal cells along the entire length of the tubules (Fig. 2A,C,E). It was localized primarily to the apical membrane of the principal cells however there appeared to be cytosolic expression in the distal portion of the Malpighian tubules (Fig. 2H) but not in the proximal region (Fig. 2G). The stellate cells expressed only P-type Na+/K+-ATPase and at a very high level relative to the neighbouring principal cells (Fig. 2A,D). However, stellate cells were observed only in the distal two thirds of each tubule (Fig. 1F, Fig. 2D) whereas the proximal third of the tubule contained only principal cells (Fig. 1F, Fig. 2B,C). Additionally, P-type Na+/K+-ATPase expression in the principal cells of the proximal region was higher relative to the distal principal cells that had very little, if any, P-type Na+/K+-ATPase expression as seen in both the whole mounts (Fig. 2B,D) and sections of the tubules (Fig. 2G,H). The proximal principal cells expressed P-type Na+/K+-ATPase on the basal membrane (Fig. 2G).
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In the anterior intestine (or anterior hindgut) of the larval A. aegypti there was no P-type Na+/K+-ATPase labeling and a very low level of V-type H+-ATPase (Fig. 2F). By contrast, the rectum had high levels of P-type Na+/K+-ATPase expression (Fig. 2F) localized to the basal region (Fig. 2I) whereas V-type H+-ATPase was not readily observed in the rectal epithelium, and there was only non-specific labeling of the fecal content (Fig. 2I).
Sections of the anal papillae showed the presence of both ATPases, with P-type Na+/K+-ATPase expressed on the basal membrane (lumen) and V-type H+-ATPase on the apical membrane facing the environment (Fig. 2J,K).
Adult female
Both ATPases were present in the adult female stomach, in the same cells
but in different regions (Fig.
3A-D). In an optical longitudinal section through the posterior
region of the stomach, the P-type Na+/K+-ATPase was seen
to be located laterally in the cell (Fig.
3A) whereas the V-type H+-ATPase was found within the
cytoplasm (Fig. 3B). Paraffin
cross sections of the stomach also indicated P-type
Na+/K+-ATPase to be located laterally in the cells, all
the way up to the apical region, in addition to being expressed on the basal
membrane. V-type H+-ATPase expression was restricted to the apical
region of the stomach epithelium (Fig.
3C,D). The entire stomach epithelium was surveyed but there were
no obvious regional differences except that perhaps there was a relatively
higher expression level for both ATPases in the posterior region of the
stomach (anterior region not shown).
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The adult Malpighian tubules exhibited the same distribution of the ATPases as described above for the larvae. The adult tubules showed high expression of the V-type H+-ATPase in the principal cells (Fig. 3F), with the highest expression on the apical membrane but cytosolic labeling was also evident (Fig. 3H, Fig. 4A,B). There was no observable V-type H+-ATPase labeling in the stellate cells (Fig. 3F). Instead stellate cells, found in the distal region, had high expression of P-type Na+/K+-ATPase relative to the adjacent principal cells (Fig. 3E). In the proximal third of the Malpighian tubules, P-type Na+/K+-ATPase labeling was higher in the principal cells relative to the distal population (Fig. 3E,G) and was localized to the basal membrane (Fig. 3H). Again, as in the larva, stellate cells (Fig. 1F, Fig. 2B,C) were not observed in the proximal thirds of the Malpighian tubules in the adults (Fig. 3G).
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RT-PCR
The genes for P-type Na+/K+-ATPase and V-type
H+-ATPase were expressed in all of the larval
(Fig. 5A) and adult tissues
(Fig. 5B).
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Malpighian tubules
Our observations of differential expression of the two ATPases in the two
cell types and the location of these cells types along the length of the
Malpighian tubules suggest a divergence in function of the principal cells in
the distal and proximal regions and a role for the stellate cells in the ion
transport processes of the distal segment.
The distally positioned principal cells exhibited high expression of V-type
H+-ATPase (Fig.
2A,E,H, Fig. 3F),
confirming previous immunohistochemical assays of both adult
(Weng et al., 2003) and larval
(Filippova et al., 1998) forms
of A. aegypti. Studies employing bafilomycin A1, a potent
inhibitor of V-type H+-ATPase, confirmed that this pump, localized
to the brush border of the apical membrane of principal cells
(Fig. 2H), is a major energy
source for the secretory processes of A. aegypti Malpighian tubules
(Beyenbach et al., 2000;
Weng et al., 2003). The V-type
H+-ATPase generates an electromotive potential by pumping protons
from the cell into the tubule lumen
(Wieczorek et al., 1999;
Beyenbach et al., 2000), thus
providing the energy to drive the secretion of cations via
Na+, K+/nH+ antiporters
(Petzel, 2000;
Weng et al., 2003). The
expression of V-type H+-ATPase throughout the cytoplasm of the
distal principal cells could be that of the dissociated V1 subunit
(Sumner et al., 1995),
cytoplasmic pools of V-type H+-ATPase being recruited to the apical
membrane, or those pumps associated with the vesicular membranes enclosing
concretion bodies. With regards to the latter possibility, concretion bodies
were previously determined to be located only in the principal cells and to
contain high concentrations of divalent cations (Ca2+,
Mg2+, Mn2+) and phosphorous
(Bradley et al., 1990).
Perhaps V-type H+-ATPase plays a role in facilitating the transport
or making available for transport (i.e. by acidification) these cations within
the concretion bodies, indicating a dual function for the H+ pump
in the distal tubule.
Several features of stellate cells observed in this study point to a role
in ion transport function. These cells, found only in the distal, secretory
region of the Malpighian tubules (Fig.
2A-E, Fig. 3E-G),
exhibited high expression of P-type Na+/K+-ATPase
(Fig. 2A,D,
Fig. 3E) and no V-type
H+-ATPase (Fig. 2E,
Fig. 3F), a pattern of
expression that contrasts with the principal cells in the same distal segment
(Fig. 2A,D,E,
Fig. 3E,F). We could not
confirm the membrane location of P-type Na+/K+-ATPase,
however, we presume this pump to be localized to the basal region of stellate
cells, where the membrane is more elaborate with extensive foldings
(Mathew and Rai, 1976;
Bradley et al., 1982;
Yu and Beyenbach, 2004) and
mitochondria (Satmary and Bradley,
1984b).
The distal location of stellate cells does suggest a secretory function,
however, the role of the Na+ pump in ion secretion in insect
Malpighian tubules, including mosquitoes, has been ambiguous because of
conflicting results utilizing the P-type Na+/K+-ATPase
inhibitor ouabain. Using adult A. aegypti Malpighian tubules, Hegarty
et al. (Hegarty et al., 1991)
reported the presence of ouabain-sensitive fluid secretion, whereas other
studies reported that ATPase activity
(Weng et al., 2003) and
membrane electrical properties (Williams
and Beyenbach, 1984) were ouabain insensitive. Interestingly,
Torrie et al. (Torrie et al.,
2004) identified an ouabain transporter (organic anion
transporting polypeptide; oatp) that colocalizes with P-type
Na+/K+-ATPase in Drosophila melanogaster
Malpighian tubules. This protein is the first step in the ouabain elimination
pathway and serves to minimize ouabain concentrations and its inhibitory
effects on P-type Na+/K+-ATPase that resides on the
basal membrane of D. melanogaster tubules
(Torrie et al., 2004).
Furthermore, highly homologous oatp genes have been identified in
both the Anopheles (Torrie et
al., 2004) and Aedes genomes (data not shown), which
indicates that these ouabain transporters are conserved within the dipterans.
If expression of this ouabain transporter protein is confirmed and found to
colocalize with P-type Na+/K+-ATPase to the stellate
cells of A. aegypti Malpighian tubules, then the ouabain
insensitivity noted in previous mosquito studies
(Weng et al., 2003;
Williams and Beyenbach, 1984)
can be explained. Our observations of A. aegypti tubules, together
with the discovery of this ouabain transport system in another dipteran
Drosophila (Torrie et al.,
2004), suggest P-type Na+/K+-ATPase and
stellate cells play a critical role in the secretory function of mosquito
Malpighian tubules.
Another intriguing aspect of stellate cell morphology that lends support
for a greater functional role was their slender, elongated, cellular
extensions that share a substantial area of contact with up to four adjacent
principal cells (Fig. 2D,E,
Fig. 3E,F). Although this was
also noted in a previous scanning electron microscopy study
(Satmary and Bradley, 1984a),
stellate cells, until now, were dismissed as being vital to secretory function
because they constitute only between 16-26% of the total cell number of
tubules (Satmary and Bradley,
1984b; Cabrero et al.,
2004), and the dimensions of their cellular extensions and contact
area with principal cells were underestimated utilizing light microscopy
(Yu and Beyenbach, 2004).
Instead, we propose that stellate cells themselves, or the interface between
stellate and principal cells, could be the site for Cl- transport,
a major aspect of the ion secretory mechanism that remains unresolved.
Currently, there are two proposed routes for transepithelial Cl-
secretion in adult Aedes Malpighian tubules: a transcellular pathway
through stellate cells and a paracellular shunt between adjacent principal
cells (Beyenbach, 2003a;
Beyenbach, 2003b;
Massaro et al., 2004;
Yu and Beyenbach, 2001;
Yu and Beyenbach, 2004).
Evidence supporting a transcellular Cl- pathway is based upon the
characterization of two types of Cl- channels on the apical
membrane of stellate cells from adult A. aegypti tubules
(O'Connor and Beyenbach, 2001)
and, more recently, leucokinin receptors localized to the stellate cell
population in adult Anopheles gambiae tubules
(Radford et al., 2004).
Previous work had confirmed that Cl- secretion is stimulated by
leucokinin in A. aegypti tubules
(Pannabecker et al., 1993;
Yu and Beyenbach, 2001;
Yu and Beyenbach, 2004),
however, the effect of leucokinin on these Cl- channels in stellate
cells has not been examined so as to confirm whether this is the primary site
of Cl- secretion. Our observation of high P-type
Na+/K+-ATPase expression in stellate cells, in addition
to the above described features of stellate cells
(O'Connor and Beyenbach, 2001;
Radford et al., 2004), lend
support for a leucokininsensitive, Na+-driven, transcellular
Cl- transport. Obviously, a basal Cl- transport
mechanism needs to be identified in the stellate cell. One possible candidate
is the bumetanidesensitive Na+/K+/2Cl-
cotransporter, which has been electrophysiologically characterized to the
basal aspect of principal cells in both A. aegypti
(Hegarty et al., 1991;
Scott et al., 2004) and
Drosophila Malpighian tubules
(Ianowski and O'Donnell,
2004). Note that in both cases, it is thought that Cl-
is recycled back to the hemolymph via Cl- channels. A
second candidate is a Na+-driven
Cl-/HCO3- exchanger (NDAE1) that colocalizes
with P-type Na+/K+-ATPase to the basal membrane of
D. melanogaster Malpighian tubules
(Sciortino et al., 2001).
Evidence for a paracellular Cl- shunt in adult Aedes
Malpighian tubules comes from transepithelial diffusional Cl-
potentials that imply a single barrier for the Cl- shunt, such as a
septate junction (Pannabecker et al.,
1993; Yu and Beyenbach,
2001; Yu and Beyenbach,
2002; Yu and Beyenbach,
2004) and leucokinin-stimulated Cl- conductance in the
distal portion of adult A. aegypti tubules that is independent of the
presence of stellate cells, thus negating a role for stellate cells in
Cl- conductance (Yu and
Beyenbach, 2004). With regards to the latter study, we assert that
it would be extremely difficult, if not impossible, to isolate a segment of
the distal tubule without some portion of one or more stellate cells being
present because of their uniform distribution thoroughout the distal region
and their long, cellular extensions that interdigitate between the principal
cells. Thus there is uncertainty as to whether the transcellular stellate cell
route or a principal-stellate cell paracellular route for the Cl-
secretion was functioning in those segments believed to be devoid of stellate
cells. An examination of the ultrastructure of the principal-principal cell
and principal-stellate cell paracellular regions and the effect of leucokinin
on these paracellular junctions may help to resolve this issue. From the above
discussion, there is still much to ascertain regarding Cl-
transport, not only in adult tubules but in larval A. aegypti, which
has garnered little attention.
Evidence of functional segmentation of A. aegypti Malpighian
tubules came from the observation of the proximal segment consisting only of
principal cells (Fig. 2B,C,
Fig. 3G) that expressed V-type
H+-ATPase and P-type Na+/K+-ATPase on the
apical and basal membrane, respectively
(Fig. 2G,
Fig. 3H). Cabrero et al.
(Cabrero et al., 2004),
examining alkaline phosphatase expression in several genera of dipterans,
confirmed the absence of stellate cells in both Anopheles and
Aedes mosquito lower tubules and suggested a role in reabsorption for
this enzyme found only in this segment of dipteran Malpighian tubules. The
lower, proximal segment of both D. melanogaster and Rhodnius
prolixus has been determined to be involved in the reabsorption of
K+ from the tubule lumen
(Maddrell and Phillips, 1975;
Maddrell, 1978;
O'Donnell and Maddrell, 1995;
Haley and O'Donnell, 1997;
Rheault and O'Donnell, 2001).
Interestingly, in D. melanogaster tubules, stellate cells are found
in greatest number in the main, secretory segment whereas in the lower,
resorptive segment, there are far fewer
(Sözen et al., 1997), a
pattern we also observed in A. aegypti tubules
(Fig. 2A-E,
Fig. 3E-G). The function of the
proximal region of the mosquito tubules has yet to be studied but it is
possible that one or both ATPases could be driving the proposed resorptive
activities. Indeed, in other insect Malpighian tubules, resorption is an
energy requiring process (O'Donnell and
Maddrell, 1995; Haley and
O'Donnell, 1997), however, the specific ATPase driving resorptive
activity has yet to be identified. Evidence of V-type H+-ATPase
activity in the resorptive segment of D. melanogaster tubules comes
from the ability to acidify lumenal fluid
(O'Donnell and Maddrell,
1995). Studies employing ouabain had indicated that P-type
Na+/K+-ATPase was not playing a role in resorption in
Rhodnius tubules (Haley and
O'Donnell, 1997) but this needs to be re-examined in light of the
recent discovery of ouabain transporters in insects
(Torrie et al., 2004). Further
examination of both the morphology and physiology of Aedes Malpighian
tubules is essential to ascertain whether we can assign both secretory and
resorptive functions to these organs. Differentiation of ion regulatory
function along the length of the tubules has been well characterized in
several diverse taxa of insects [e.g. hymenopterans
(Arab and Caetano, 2002),
orthopterans (Kim and Spring,
1992), hempiterans (Maddrell
and Phillips, 1975)] including dipterans
(Dow et al., 1994;
O'Donnell and Maddrell,
1995).
Our observations, described in this section, hold true for both the larval
and adult Malpighian tubules, suggesting that there are no apparent
morphological distinctions or differences in ATPase function between the two
life stages despite having disparate osmoregulatory demands. Mosquito
Malpighian tubules do not undergo autolysis during metamorphosis
(Clements, 2000) or
morphological rearrangements via a change in cell number or
proportion of principal cells to stellate cells
(Satmary and Bradley, 1984b).
Perhaps the source of differentiation in ion transport function between larval
and adult A. aegypti is a consequence of differential modulation of
expression and/or regulation of the ATPases or the population of secondary ion
transporters (i.e. Na+, K+, Cl- channels,
antiporters and symporters) that these two ATPases energize.
Midgut
In contrast to the Malpighian tubules, the midgut differs dramatically
between the larval and adult stages both in gross morphology and location of
the two ATPases.
The gastric caeca, the eight blind-end sacks at the anterior aspect of
larval midgut, showed regional ATPase expression differences that correlate
with their physiological function as designated by their ultrastructural
characteristics (Volkman and Peters,
1989a; Volkman and Peters,
1989b; Zhuang et al.,
1999). Located in the distal third of each caecum,
ion-transporting cells are thought to absorb ions from the caecal lumen
(Volkmann and Peters, 1989b). It is here that we observed V-type
H+-ATPase on both membranes of these cells, indicating a novel
ion-transporting tissue (Fig.
1B,D). V-type H+-ATPase activity in these cells was
substantiated in the measurements of a bafilomycin-sensitive, inward alkaline,
pH gradient at the distal aspect of the gastric caecum of A. aegypti
larvae (Boudko et al., 2001).
Additionally, morphological studies reported that the apical membrane of the
ion-transporting cells possess long, slender microvilli studded with
portasomes and mitochondria extending up into each microvillus (Volkmann and
Peters, 1989a; Zhuang et al.,
1999), traits analogous to the principal cells of Malpighian
tubules and the presence of V-type H+-ATPase
(Bradley and Snyder, 1989).
The basement membrane of these cells, however, has not been examined for the
presence of portasomes (i.e. the V1 subunit of V-type
H+-ATPase), but only characterized as having extensive infoldings
and associated mitochondria (Volkman and
Peters, 1989a; Zhuang et al.,
1999). Perhaps the apical and basal V-type H+-ATPase
function under different conditions such as when the ion or pH level of
ingested water varies. Indirect support for this comes from ultrastructural
changes in the ion-transporting cells with increased water salinity,
specifically the reduction in apical microvilli length, accumulation of
mitochondria in the basal region and enhancement of basal channels (Volkmann
and Peters, 1989b). This may also suggest a reversal in the direction of ion
and water movement from absorption to secretion with high salinity water being
ingested.
The resorbing/secretory cells that comprise the proximal two thirds of each
caecum of larval A. aegypti (Volkmann and Peters, 1989a) had high
expression of P-type Na+/K+-ATPase on the basal membrane
and V-type H+-ATPase on the apical membrane facing the lumen
(Fig. 1B-D). These cells have
extensive infoldings of both the basal and apical membranes and each are
populated with mitochondria. However, these organelles do not reside within
the apical microvilli as in the ion-transporting cells (Volkmann and Peters,
1989a). It is conceivable that one or both of the ATPases are involved in the
nutrient resorption activity of these cells as amino acid and glucose uptake
in insects can be Na+ or H+ driven
(Hediger, 1994;
Harvey and Wieczorek, 1997;
Liu et al., 2003).
Interestingly, when external salinity is varied, these cells, like the
ion-transporting cells, alter their ultrastructure suggesting these
ion-dependent nutrient transport systems could be modulated (Volkmann and
Peters, 1989b).
Our present examination of the main portion of the larval midgut found
variation in the membrane location of both ATPases. This first ever look at
P-type Na+/K+-ATPase expression in A. aegypti
larval midgut revealed high expression of this pump throughout this tissue
(Fig. 1E-I). As a consequence,
our findings introduce further complexity to the current osmoregulatory and
nutrient absorption models for this tissue. Previous examinations of A.
aegypti larval midgut (Filippova et
al., 1998; Gill et al.,
1998; Zhuang et al.,
1999; Boudko et al.,
2001) had focused on the role of V-type H+-ATPase in
the alkalinization process and assumed that it was the sole source of proximal
energy to drive ion and nutrient transport. The main reason for this was that
the model for larval mosquito midgut alkalinity was adopted from the
lepidopteran midgut, which had previously been documented to be devoid of
P-type Na+/K+-ATPase activity
(Wieczorek et al., 1999). As
we have stated above, P-type Na+/K+-ATPase function
needs to be reconsidered in light of the recent discovery of ouabain
transporters in dipterans (Torrie et al.,
2004).
V-type H+-ATPase expression in the larval midgut was similar to
patterns previously examined (Filippova et
al., 1998) including studies utilizing an antibody specific to the
E subunit of the V-type H+-ATPase, with localization patterns
correlating with the presence of portasomes (V1 subunit of V-type
H+-ATPase) (Zhuang et al.,
1999), bafilomycin-sensitive pH gradients
(Boudko et al., 2001) and
transepithelial potentials (TEP) (Clark et
al., 1999). The highest V-type H+-ATPase expression was
evident in the mid-anterior region (Fig.
1E) and was localized to the basal membrane
(Fig. 1G,H) where portasomes
line the extensive infoldings of this membrane
(Zhuang et al., 1999). It is
in this segment that lumenal pH is most alkaline (pH >10)
(Zhuang et al., 1999;
Boudko et al., 2001), and the
TEP is lumen-negative with the basement membrane hyperpolarized
(Clark et al., 2000). All of
this evidence points to the extrusion of protons into the hemolymph
via a basal V-type H+-ATPase. In the posterior midgut, the
V-type H+-ATPase switched to an apical location
(Fig. 1I) and is supported by
previous findings of apical membrane hyperpolarization, lumen positive TEP
(Clark et al., 1999) and
colocalization of the E subunit of V-type H+-ATPase and portasomes
to this membrane (Zhuang et al.,
1999).
In the very anterior of the larval midgut, P-type
Na+/K+-ATPase localized to the basal membrane with no
V-type H+-ATPase expression in these cells
(Fig. 1E,G). This particular
region has not been described with regards to its ultrastructure or
physiology, so a potential role for the high density of basal P-type
Na+/K+-ATPase cannot be put forth at this time.
Recently, Onken et al. (Onken et al.,
2004) reported an ouabain-sensitive transepithelial voltage in the
anterior midgut region of larval A. aegypti and proposed that P-type
Na+/K+-ATPase was localized to the basal membrane.
Perhaps it was this very anterior region that Onken et al.
(Onken et al., 2004)
characterized because this section lacks V-type H+-ATPase. Further
along the anterior midgut segment (Fig.
1H) the P-type Na+/K+-ATPase was located
apically, then basally in the posterior region
(Fig. 1F,I).
This intriguing flipping pattern of the two ATPases between the apical and
basal membranes down the length of the gut suggests that the activity of the
two pumps could be coordinated as it is in other ion transporting epithelia
(Ehrenfeld and Klein, 1997).
For instance, it was suggested that the source of protons for the basal V-type
H+-ATPase of the anterior midgut could be the gut lumen and that
transport into the cell could occur through an apically located
K+/nH+ antiporter
(Boudko et al., 2001). If this
configuration is present, then the apical P-type
Na+/K+-ATPase could function to maintain the diffusive
outward K+ gradient, thereby driving K+/H+
exchange on this membrane. In the posterior midgut, the basal P-type
Na+/K+-ATPase could power Na+ absorption as
A. aegypti larvae typically reside in Na+-poor
environments and may rely upon diet as a Na+ source. Na+
absorption could occur across the apical membrane through a Na+
channel coupled to the V-type H+-ATPase with transport across the
basal membrane via Na+/K+-ATPase
(Ehrenfeld and Klein, 1997).
This inward Na+ gradient could also aid in the absorption of amino
acids. An amino acid transporter (KAAT1) was cloned and characterized in
Manduca and was found to accept both Na+ and K+
as cosubstrates (Liu et al.,
2003). To truly characterize ATPase function in the larval midgut,
it will be necessary to know the ionic concentrations of the lumen and midgut
cells in the anterior and posterior regions. This will then help to describe
the environment in which P-type Na+/K+-ATPase and V-type
H+-ATPase are functioning when expressed on either membrane. This,
in addition to further physiological characterization of ion transport in the
midgut, will help to clarify coordinated or independent functioning of the two
ATPases.
In the midgut of the adult female (also referred to as the stomach), P-type
Na+/K+-ATPase and V-type H+-ATPase were
expressed on the basolateral and apical membrane respectively
(Fig. 3A-D) with perhaps,
qualitatively, higher levels of expression of both ATPases in the posterior
region (comparison not shown). This pattern correlates with the morphological
features of the posterior region that are indicative of ion and water
transport function (Billingsley,
1990). The posterior midgut is also the region where the blood
meal, taken by a female mosquito, is moved to
(Nation, 2002). Columnar
cells, the predominant cell type in the midgut, possess very elaborate apical
and basolateral membrane systems with the latter forming a basal labyrinth
consisting of extensive, deep infoldings laden with mitochondria
(Clements, 2000). It is within
this region we believe P-type Na+/K+-ATPase is located
because of the intense and expansive labeling of this pump on the lateral
aspect (Fig. 3A,C) and
throughout the basal region (Fig.
3C,D). Additional evidence of P-type
Na+/K+-ATPase was provided by the finding that more than
84% of ATPase activity in adult mosquito stomach is ouabain sensitive
(MacVicker et al., 1993). This
ATPase may serve a role in the absorption of ions and water across the adult
midgut in a manner similar to that described for solute-coupled water
transport in the mammalian small intestine
(Larsen et al., 2002). In this
model, the laterally located P-type Na+/K+-ATPase drives
both inter- and transcellular water and ion absorption via the recirculation
of Na+ through a basolateral
Na+/K+/2Cl- cotransporter. Interestingly,
expression of the P-type Na+/K+-ATPase gene in the
stomach of adult female A. aegypti decreased following a blood meal
(Sanders et al., 2003),
perhaps as a means to attenuate the proposed solute-coupled water transport,
thus avoiding excessive ion and water influx across the gut. By contrast,
V-type H+-ATPase gene expression in the stomach rapidly increased
following a blood meal. Concurrent with the H+ pump expression
pattern was the upregulation of amino acid and sugar transporter genes
(Sanders et al., 2003), which
together, may suggest a H+-driven nutrient absorption mechanism in
the adult female stomach. Examination of protein expression and activity of
these two ATPases and the secondary transporters coupled to these ATPases is
essential to help elucidate their role in the diuresis response of adult
mosquitoes.
Hindgut and larval anal papillae
Although expression of P-type Na+/K+-ATPase and
V-type H+-ATPase genes was evident in the larval rectum
(Fig. 5A), only P-type
Na+/K+-ATPase protein labeling was observed
(Fig. 2F,I), suggesting that it
is the primary pump energizing the resorption of Na+, K+
and Cl- from the urine, as previous in vitro and in
vivo investigations have detailed
(Bradley, 1987;
Bradley, 1994). Our finding of
high expression of P-type Na+/K+-ATPase on the basal
membrane of the rectum (Fig.
2F,I) is in agreement with other insect studies. P-type
Na+/K+-ATPase has been found in the rectum in D.
melanogaster using the same 5 antibody
(Lebovitz et al., 1989) and
also by ouabain-inhibitable sensitivity assays in the locust rectum
(Peacock, 1981). The
basolateral region of the rectal epithelium of larval A. aegypti
consists of an elaborate labyrinth that appears to extend almost to the apical
region (Clements, 2000). Based
on the intense P-type Na+/K+-ATPase labeling that
appears to occupy the basal two-thirds of the rectal cells
(Fig. 2I), we propose this pump
is residing on these basal infoldings. However, the lack of V-type
H+-ATPase expression in the rectal epithelium of larval A.
aegypti (Fig. 2F,I)
conflicts with other findings (Filippova
et al., 1998) where, using the same V-type H+-ATPase
antibody, high expression in the rectum of this species was reported. However,
only whole-mount specimens where examined in the previous study and hence the
non-specific V-type H+-ATPase labeling of the rectal contents was
not detected as it was in the paraffin sections of this present study
(Fig. 2I). Although the
ultrastructure of the Aedes larval rectum has yet to been examined,
in Culex mosquito larvae, the apical membrane of the rectal cells is
extensive and studded with portasomes
(Bradley, 1987) suggesting that
the V-type H+-ATPase should be there.
We successfully determined that V-type H+-ATPase and P-type
Na+/K+-ATPase proteins immunolocalize to the apical and
basal membranes, respectively, in the anal papillae of larval A.
aegypti (Fig. 2J,K).
Earlier studies had pinpointed the anal papillae as the predominant organs for
Na+ and Cl- uptake in freshwater mosquito larvae
(Wigglesworth, 1938;
Treherne, 1954;
Stobbart, 1965), however,
these four, cuticle-lined, saclike organs have been troublesome for the
examination of ion transport processes because of their design
(Bradley, 1994). More recently,
with the use of self-referencing ion-selective (SeRIS) microelectrodes, Donini
and O'Donnell (Donini and O'Donnell,
2005) confirmed that the anal papillae of larval A.
aegypti serve as the major site for Na+, Cl- and
K+ uptake. With regards to the role of ATPase, Patrick et al.
(Patrick et al., 2002)
characterized in vivo Na+ and Cl- uptake in
A. aegypti larvae and found that V-type H+-ATPase was
involved in both transport processes, but in a novel way. The external
application of bafilomycin A1, a specific antagonist of V-type
H+-ATPase, resulted in a stimulation of Cl- uptake
(Patrick et al., 2002), a
pattern that departs from all other Cl- absorptive processes
examined (Fenwick et al.,
1999; Phillips et al.,
1996) and intimates a novel H+-dependent Cl-
uptake mechanism (i.e. Cl- channel). In the same experiment,
bafilomycin inhibited Na+ uptake, indicating an apical V-type
H+-ATPase/Na+ channel moiety as described in other
freshwater animals, where the extrusion of protons sets up the inward
electrodiffusive gradient for Na+
(Fenwick et al., 1999;
Ehrenfeld and Klein, 1997).
Our present finding of apical V-type H+-ATPase expression
(Fig. 2J,K), along with reports
of the apical lamella being studded with portasomes and associated with
mitochondria (Sohal and Copeland,
1966; Meredith and Phillips,
1973; Garrett and Bradley,
1984) bolsters the findings of Patrick et al.
(Patrick et al., 2002). The
role of the basal Na+/K+-ATPase in anal papillae remains
to be probed but could serve as the entry step of Na+ into the
hemolymph. The extensive, deep basal infoldings and tightly associated
mitochondria (Sohal and Copeland,
1966; Meredith and Phillips,
1973; Garrett and Bradley,
1984) is a feature shared with the larval rectum (see above) and
indicative of an ion transport role. One way in which this ion transporting
tissue differs from others in the larva is that it is syncytial, meaning it
lacks a lateral cell wall. Perhaps this unusual morphology gives rise to the
novel ion transporter configurations (i.e. Cl- transport) noted
previously (Patrick et al.,
2002).
To date, only morphological studies have been performed on the adult
mosquito hindgut. Our study is the first to examine specific ion transport
proteins in these epithelia. The patterns of basolateral P-type
Na+/K+-ATPase and apical V-type H+-ATPase
localization in the adult hindgut (Fig.
4), in addition to previous studies
(Hopkins, 1967;
Tongu et al., 1969) infer that
both the anterior hindgut and rectal pads are sites of ion and water movement.
The rectum of adult A. aegypti consists of a thin rectal sac in which
six (female) or four (male) rectal pads
(Clements, 2000) protrude into
the lumen (Fig. 4A-D). By
contrast, the anterior hindgut is a thicker epithelium
(Fig. 4A,B,E,F) relative to the
rectal sac (Fig. 4A). Both the
rectal pads and the anterior hindgut possess a well-developed basolateral
labyrinth populated with mitochondria, a trait shared with the larval rectum
and anal papillae (Clements,
2000). Following a blood meal, the most notable changes occurred
in the basal and lateral membrane systems of the rectal pads, intimating
enhanced ion and water absorption activity
(Hopkins, 1967). In
Fig. 4C, the intense P-type
Na+/K+-ATPase labeling on these basolateral infoldings
of the rectal pads indicate that the Na+ pump must be driving these
absorptive processes following a blood meal. The apical membrane of the
anterior hindgut contains elaborate infoldings with mitochondria
(Tongu et al., 1969) and was
found to highly express the H+ pump
(Fig. 4E,F). By contrast,
Hopkins (Hopkins, 1967),
reported that the apical membrane of the rectal pads is not elaborately
infolded. We observed only a thin layer of V-type H+-ATPase
expression in this region (Fig.
4C,D) thus confirming that this membrane is not extensive.
Additional studies are necessary to determine what role these organs and two
ATPases serve in the adult mosquito osmoregulatory activities.
Conclusion
We have established the presence of P-type
Na+/K+-ATPase and V-type H+-ATPase in the key
osmoregulatory tissues of larval and adult A. aegypti. The patterns
of protein expression, as determined through immunolocalization techniques,
suggest that both ATPases are contributing to solute transport, specificially
by providing the proximal energy, in the form of favourable electrochemical
gradients, to move ions and organic solutes across membranes. Now, we can
identify the specific roles of P-type Na+/K+-ATPase and
V-type H+-ATPase in the two life stages of the A. aegypti
by monitoring the gene and protein expression profiles of these two pumps
during the mosquito's response to physiological challenges such as varying the
chemical composition of the larva's watery environment or a blood meal taken
by the adult female. These studies will then provide information on how the
ATPases are regulated.
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