A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK
Author for correspondence (e-mail:
j.a.t.dow{at}bio.gla.ac.uk)
Accepted 18 June 2004
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Summary |
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Cell counts are also provided for each species. As in Drosophila, stellate cells are not found in the lower tubule domain of Anopheles or Aedes tubules, confirming the unique genetic identity of this domain. As previously reported, we failed to find stellate cells in Schistocerca but, remarkably, also failed to find them in Glossina, the dipteran most closely related to Drosophila. The orthodoxy that stellate cells are unique to, and general among, Diptera may thus require revision.
Key words: Drosophila, Aedes, Anopheles, Glossina, Schistocerca, Diptera, alkaline phosphatase, stellate cell.
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Introduction |
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In insects, functional ALP has been shown to exist in Leptinotarsa
decemlineata (Colorado potato beetle;
Yi and Adams, 2001),
Bombyx mori (silkworm) gut (Eguchi
et al., 1990
), Bemisia tabaci (whitefly) salivary glands
(Funk, 2001
), Drosophila
melanogaster brain (Yang et al.,
2000
) and several mosquito species
(Houk and Hardy, 1984
;
Igbokwe and Mills, 1982
).
However, given their role in secretory function, ALPs have also been studied
in insect Malpighian tubules, which are critical osmoregulatory and secretory
organs in insects; ALP expression has been documented in L.
decemlineata and D. melanogaster
(Yang et al., 2000
) tubules.
In particular, the D. melanogaster tubule is a genetically accessible
transporting epithelium (Dow and Davies,
2003
) and is thus a useful model in which to study the functional
role of ALP in vivo.
Previous work in D. melanogaster has shown that tubule ALP
expression is confined to cells in the lower segment
(Sozen et al., 1997;
Yang et al., 2000
), which
plays a role in fluid re-absorption
(O'Donnell and Maddrell,
1995
). Furthermore, a P-element mutation in the ALP gene,
Aph-4, causes misexpression of Aph-4 in the tubules; expression is
substantially reduced in the lower tubule but increased in the main segment.
This misexpression results in reduced basal and stimulated fluid secretion by
the tubules. Thus, ALP is likely to have a significant - although as yet
unexplained - role in epithelial transport in D. melanogaster.
Insect Malpighian tubules are important to insect life and therefore
constitute important targets for population control. The power of D.
melanogaster as a genetic model organism has allowed significant steps in
understanding tubule function (Dow and Davies,
2001,
2003
), which can then be
applied to those insect species with less-developed genomic resources but
greater economic or medical significance; for example, tsetse fly or mosquito.
Thus, we have begun to apply knowledge of tubule function in D.
melanogaster to those of insect vector species with a view to advancing
understanding of tubule physiology in the context of specific cell types and
tubule regions in these animals. The species selected for the present study,
in addition to D. melanogaster, are Anopheles stephensi (a
malarial mosquito), Aedes aegypti (a mosquito vector for dengue
fever) and Glossina morsitans (a vector for African sleeping
sickness). Together, these provide a wide phylogenetic spread through the
Diptera, the largest insect order. Additionally, we selected the locust
Schistocerca gregaria as an out-group from the exopterygote order
Orthoptera.
We show here that tubules from dipteran species - An. stephensi, Ae. aegypti and G. morsitans - share identical domains of ALP expression to D. melanogaster. However, tubules from Schistocerca gregaria do not show any significant staining for ALP. Thus, defined ALP expression in dipteran tubules may be an important feature of fluid transport mechanisms by these tubules and may constitute important control elements for fluid secretion/absorptive pathways.
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Materials and methods |
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All insects were cold-anaesthetised and decapitated prior to dissection to isolate intact tubules.
Histochemistry for alkaline phosphatase activity
The methods for staining of alkaline phosphate in tubules was essentially
that of Yang et al. (2000).
Briefly, intact tubules were dissected into wells containing
phosphate-buffered saline (PBS) and treated with 4% (v/v) paraformaldehyde for
20 min. Tubules were washed for 3x10 min in PBS/0.1% Triton and then for
2x2 min in PBS. Tubules were incubated for 10-30 min with 75 mg
ml-1 4-nitroblue tetrazolium chloride (NBT)/50 mg ml-1
5-bromo-4-chloride-3-indolyl-phosphate (BCIP) in dimethylfluoride (DMF) and
used at 4.5 µl of NBT and 3.5 µl BCIP per 1 ml DIG kit detection buffer,
pH 9.5 (Boehringer, Bracknell, UK). Control tubules were processed in the same
way with the exception of NBT/BCIP addition. Tubules were washed for 3x2
min in PBS, viewed under a standard microscope and photographed with a digital
camera.
Where required, tubules were stained for ALP activity and with 4',6'-diamidino-2-phenylindole hydrochloride (DAPI; Sigma-Aldrich, Gillingham, UK). For these experiments, 1 µg ml-1 DAPI was added in PBS to ALP-stained tubules after the final 3x2 min PBS washes (see above) for 2.5 min. Tubules were washed in PBS for 3x2 min.
For all protocols, prior to mounting on slides, tubules were washed with glycerol as follows: 2x20% glycerol/PBS (v/v); 2x50% glycerol/PBS (v/v); 2x80% glycerol/PBS (v/v). Samples were viewed with the Axiocam imaging system (Zeiss, Welwyn Garden City, UK).
Statistical analysis
Where appropriate, the significance of differences was tested with
Student's t-test (two-tailed), assuming unequal variances and taking
the critical level to be P<0.05.
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Results |
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Amongst the dipteran species tested, cell number per mm of tubule was similar between the mosquitoes and G. morsitans. D. melanogaster, however, has vastly more cells per mm of tubule: nearly 80 in D. melanogaster versus 11-17 in the other Diptera.
We took the opportunity to perform a systematic cell count of each tubule
in the two mosquito species (Fig.
1). This shows that while each of the tubules differs in cell
count from the others, this is mainly attributable to differences in principal
cell number. Both stellate cells and the numbers of cells in the ALP domain
are almost invariant between tubules. It is also worth noting that, as in
D. melanogaster, cell numbers are almost invariant. These data thus
usefully show that the precision with which the D. melanogaster
tubule is specified (Sozen et al.,
1997) is not unique to this genetic model organism.
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Use of the locust as an out-group in this study shows that the notable differences between the S. gregaria tubule and those of the Diptera are numbers of tubules per insect and the lack of stellate cells (compared with D. melanogaster, Ae. aegypti and An. stephensi).
Alkaline phosphatase activity is confined to the lower tubule in dipteran insects
Previous work has shown that ALP expression in D. melanogaster
tubules is confined to the lower tubule and precisely matches the lower-main
segment boundary (Yang et al.,
2000). On close examination, combined with DAPI counter-staining
of nuclei, it is clear that ALP is concentrated in the apical membrane
(Fig. 2D).
While the genetic determination of tubule sub-regions is currently unknown in other dipteran species, we show that in Ae. aegypti, An. stephensi and G. morsitans, staining is confined to the lower tubule (Figs 3, 4, 5). Interestingly, in spite of the different lengths of mosquito tubules, expression of ALP is observed in the lower domain to the same extent in all tubules in both Ae. aegypti and An. stephensi (Fig. 1).
In tubules of the out-group species, S. gregaria, no staining for ALP was ever observed (Fig. 6). Incubation conditions were varied to maximise the chances of observing ALP staining, including increasing staining times to up to 24 h. However, specific staining was not noted under any experimental condition.
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Alkaline phosphatase staining in the lower domain involves precise numbers of cells
Counterstaining for cell nuclei and ALP allowed the determination of cell
numbers within the tubule lower domains for each species (Figs
2D,
3D,
4D,E,
5F).
Table 2 shows the results for
such analysis. Consistent with the fact that Ae. aegypti and An.
stephensi tubules have fewer cells than either D. melanogaster
or G. morsitans, there are fewer cells in the ALP domain of both
mosquito species. Interestingly, while the absolute number of cells in the ALP
domain may vary between species, the percentage of all tubule cells localised
to the ALP domain in the dipteran species studied is virtually identical
(10). Thus, the distribution and function of the cells in this lower
domain must be specified by the same genetic determinants in each of the
dipteran species studied here.
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Furthermore, the relative numbers of stellate cells in D. melanogaster,
Ae. aegypti and An. stephensi are the same: 18%. This further
supports the view that the genetic determinants that set tubule domains are
conserved across the Diptera.
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Discussion |
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We have shown for the first time that ALP activity marks a conserved lower
tubule domain of dipteran insects. While it is impossible to assert absolutely
that this tightly conserved expression domain represents a conserved vital
function, the closeness of conservation across even a wide range of dipteran
taxa suggests strongly that there is a functional role required of the lower
10% of dipteran tubules. It will be interesting (though technically very
demanding) to establish in the future whether the reabsorptive function of
this domain is also conserved beyond Drosophila. In D.
melanogaster, the ALP expression pattern is identical to that marking the
lower tubule domain by enhancer trapping
(Yang et al., 2000). Data in
Table 2 also show that the
proportion of cells in the ALP domain compared with overall cell number in the
tubule is similar in the dipteran species tested. However, the tsetse tubule
can be differentiated from those of the other Diptera: it lacks stellate
cells. To our knowledge, this is the first time that stellate cells have been
found to be absent in a dipteran.
A mutation in the D. melanogaster ALP gene results in reduced
basal rates of fluid transport (Yang et
al., 2000) without affecting stimulated transport via the
neuropeptides CAP2b (Davies et
al., 1995
) or drosokinin
(Terhzaz et al., 1999
). Thus,
this suggests that ALP has a specific role in maintaining resting rates of
transport via cellular events in the lower domain. Given that the
lower domain has been shown to be involved in fluid reabsorption
(O'Donnell and Maddrell,
1995
), the possibility exists that ALP influences the overall rate
of fluid transport by controlling fluid reabsorption. By extension, this
situation may also exist in the mosquito and tsetse tubule. Developmental work
in Schistocerca americana has demonstrated anchoring of epithelial
ALP to the plasma membrane by glycosyl-phosphatidylinositol and involvement of
phospholipase C in release of ALP from the anchoring sites
(Chang et al., 1993
). Given
that fluid transport in D. melanogaster tubules is under control by
several signalling pathways (Dow and
Davies, 2001
), including that involving phospholipase C (PLC;
Pollock et al., 2003
), it is
entirely possible that signalling processes are an important control mechanism
for fluid reabsorption involving ALP action.
In this work, we show that the ALP/lower domain is conserved in dipteran
species but not in an out-group, S. gregaria. Does S.
gregaria express ALP at all? Published work has shown that both fat body
(George and Eapen, 1959) and
alimentary canal (Navqi, 1981
)
contain ALP. However, expression in tubules has not been documented. Thus, the
important role for ALP in the lower tubule may not be a feature of orthopteran
insects.
There are also intriguing comparative aspects to our study
(Fig. 7). The tsetse fly is the
most closely related to D. melanogaster; consistent with this, it has
four tubules, arranged in two pairs, each sharing a common ureter. However, it
lacks stellate cells, even though these are found in the much more distantly
related mosquito species, although they each have five tubules that do not
have shared ureters. Stellate cells have also been demonstrated in a species
of intermediate distance, Calliphora erythrocephala
(Berridge and Oschman, 1969).
It is thus reasonable to suppose that the unique lifestyle of G.
morsitans (it is an obligate blood-feeder and gives birth to live young)
has led to secondary loss of the dipteran stellate cell. There may thus be
useful discrimination built into these simple functional morphological
techniques; it will be interesting to extend them to other insects in the
future.
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Acknowledgments |
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Footnotes |
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