pH tolerances and regulatory abilities of freshwater and euryhaline Aedine mosquito larvae
1 Department of Biological Sciences, Indiana University, South Bend, IN
46634-1700 USA
2 Department of Ecology and Evolutionary Biology, Osborn Memorial
Laboratory, Yale University, New Haven, CT 06520, USA
* Author for correspondence (e-mail: tclark2{at}iusb.edu)
Accepted 7 April 2004
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Summary |
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Key words: mosquito larvae, pH regulation, pH acclimation, life history, Aedes aegypti, Ochlerotatus taeniorhynchus
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Introduction |
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Despite the great range of ambient pH values tolerated by larval mosquitoes
in nature and in the laboratory, we have almost no information about the
effects of pH on larval growth and development or about regulation of
hemolymph pH in these animals. The predominant physiological challenges of
life in acidic water are Ca2+ regulation, Na+
regulation, and mobilization of toxic metals such as aluminum from the
substrate (Wiederholm, 1984).
Most aquatic insects that have been examined are able to maintain relatively
constant hemolymph pH when exposed to acid waters. This is apparently achieved
using ion exchange mechanisms, especially Na+/H+
exchangers, to move acid/base equivalents. Because of this, the mechanisms of
low pH toxicity appear to relate more strongly to disruption of general ionic
balance, especially Na+, than to failure of pH homeostasis per
se (Havas, 1981
). Low pH
is thus generally more harmful in oligotrophic, low-ionic strength water,
where ions involved in coupled transport of acid or base equivalents are
limited (Vangenechten et al.,
1989
). Survival in alkaline conditions has received less
attention, but in larval mosquitoes it is known to involve
Cl/HCO3 exchange occurring in
the rectum (Stobbart, 1971
;
Strange et al., 1982
,
1984
;
Strange and Phillips,
1985
).
The present work investigates for the first time the effects of ambient pH
on growth and development of larval mosquitoes. We compare the effects of
ambient pH on two mosquito species with very different saline tolerances
within the subfamily Aedini, Aedes aegypti (L.) and Ochlerotatus
taeniorhynchus (Wiedemann) (these species were considered congeneric
until recently; Reinert,
2000). Aedes aegypti is an obligately freshwater species
that inhabits open containers such as tin cans and discarded tires, and is
unable to survive in waters of salinity greater than about 40% seawater (14 g
l1). The euryhaline O. taeniorhynchus can complete
development in waters ranging from fresh to those more concentrated than
full-strength seawater (35 g l1) (for the salinity
tolerances of the larvae used in these experiments, see
Clark et al., 2004
). Because of
the known interactions between pH regulation and ionoregulation, we were
interested to ascertain whether the distinct saline tolerances of these two
species were correlated with differences in pH regulatory abilities. In
addition, we wished to determine whether mosquito larvae possess the capacity
for acclimatory responses to pH, and whether there are physiological
trade-offs inherent in the mechanisms used to deal with acid or base
loads.
Data presented here demonstrate that (1) O. taeniorhynchus and
A. aegypti have very similar pH tolerances, completing larval
development in buffered waters ranging from pH 4 to pH 11 in the laboratory.
(2) Larvae of both species are highly effective pH regulators, maintaining
hemolymph pH within narrow limits across the entire tolerable pH range. (3)
The effects of pH on larval growth and development are quite similar in the
two species, and are minor in comparison with the influences of sex, and
species. (4) Further studies in A. aegypti alone showed that the
ionic composition of the water (3.5 g l1 NaCl, 3.5 g
l1 sea salt, no added salt) had no effect on the range of pH
tolerated. (5) Aedes aegypti can acclimate to either acidic or basic
conditions without interfering with survival at the other extreme of pH. This
demonstrates that the mechanisms used to regulate body pH in acidic and
alkaline conditions are not mutually exclusive and thus may be physically or
temporally separated, as in the acid- and base-secreting intercalated cells of
the mammalian nephron (Brown et al.,
1992).
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Materials and methods |
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Solutions
Three different rearing solutions were used. All rearing solutions
contained 2.5 mOsm l1 Trizma base
(Tris[hydroxymethyl]aminomethane), and 2.5 mOsm l1 Hepes
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid]), and were adjusted to the appropriate pH using HCl or NaOH. One rearing
solution, subsequently referred to as sea salt, contained in addition to these
buffers 3.5 g l1 (83 mOsm l1) artificial
sea salt (Instant Ocean; Aquarium Systems, Mentor, Ohio, USA). A second
rearing solution, referred to hence as NaCl, contained in addition to the
buffers 3.5 g l1 NaCl (59.9 mOsm l1),
while in a third rearing solution the added ions were limited to the NaOH or
HCl used to adjust the pH and those present in the food. pH was determined
using a Perphect LogR meter model 330 (Orion, Beverly, MA, USA), with an Orion
Perphect gel-epoxy triode electrode.
pH tolerance and the influence of pH on life history parameters
Larvae were reared as described above, in batches of 20 larvae per 50 ml,
until all larvae had died or pupated. Live pupae were collected each morning,
blotted dry, and weighed to the nearest 0.01 mg using a high precision
analytical balance (Mettler Toledo AX 205 Deltarange; Columbus, OH, USA). Dry
mass was determined after drying at 65°C for 24 h. Masses of dead larvae
or pupae were not determined (mosquitoes have pupae that are highly mobile,
and dead pupae can be readily distinguished by their failure to remain within
the water column, swim to the surface to obtain air, or respond to the
presence of the investigator).
Transfer experiments
Larvae were reared for 3 days in buffered, 3.5 g l1 NaCl
solution, at pH 4, 7 or 11. Larvae were acclimated in batches of 10 larvae,
and after 3 days of acclimation survivors of each batch were either maintained
in the rearing pH (4 to 4 or 11 to 11) or transferred to another pH (7 to 12,
7 to 3, 4 to 11, 4 to 3, 11 to 4, 11 to 12), with the first number
representing the acclimation pH and the second the pH in which they completed
development. Water was changed and larvae were fed each day. Live pupae
appearing during the previous 24 h werecollected each morning, weighed and
sexed.
Measurement of hemolymph pH
pH electrodes were made from double-barreled borosilicate omega dot
capillary tubing (1.5 mm o.d., 0.75 mm i.d.; FHC, Brunswick, ME, USA). The
glass tubing was washed with nitric acid, then with nanopure water (R
>17 M) and oven dried. Electrodes were pulled on a Kopf (Tujunga,
CA, USA) Model 720 Needle/Pipette puller. The tips to contain pH resin were
sylanized using 5% dimethyldichlorosilane (Sylon CT; Supelco, Bellefonte, PA,
USA), and dried with gentle heat on a hot plate. The tip of the silanized
barrel was filled with resin (Hydrogen Ionophore 1; Sigma, St Louis, MO, USA)
and the barrel was backfilled with 0.5 mol l1 KCl. The
reference (ground) barrel was also filled with 0.5 mol l1
KCl. The electrode tip was broken to reduce electrical resistance. Ag/AgCl
electrodes were inserted into each barrel.
Larvae reared in buffered, 3.5 g l1 NaCl solutions of different pH (see above) were rinsed in deionized water, blotted dry, and torn open on Parafilm using fine forceps. A pH microelectrode with grounded reference barrel was then immediately inserted into the drop of hemolymph. The voltage was determined using a high impedence amplifier (Iso-DAM8; World Precision Instruments; Sarasota, FL, USA), coupled to Sable Systems Data Acquisitions System (Sable Systems, Henderson, NV, USA). Voltage was converted to pH by reference to a standard curve.
Statistical analyses
Mortality rates of the two species were compared using Fisher's exact test
(SAS frequency procedure; SAS Institute Inc. 1997). Mortality across salt
types within pH were investigated using single factor analysis of variance
(ANOVA). The dependence of larval hemolymph pH on rearing pH was analyzed for
each species using a one-factor analysis of variance, with rearing pH as a
categorical predictor (Microsoft Excel 2002). Effects of pH on life history
parameters were modeled using mixed linear models (SAS mixed procedure; SAS
Institute Inc. 1997). Models testing for effects of species, sex, pH and their
interactions, included as categorical variables, were further explored with
separate models fitted for each species. Significant pHxsex interaction
effects were further explored using sub-models in which each sex was
considered separately. Acclimation experiments were analyzed using Student's
t-tests in Microsoft Excel.
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Results |
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Hemolymph pH
Larvae of both species reared in waters ranging from pH 4 to pH 11 show
relatively constant hemolymph pH values, measuring above 7.5 across the entire
pH range (Fig. 1). Hemolymph pH
values of A. aegypti and O. taeniorhynchus are comparable
(pH 7.7) in animals reared at pH 11. Hemolymph pH of O.
taeniorhynchus is independent of ambient pH (P>0.9; single
factor ANOVA), measuring around pH 7.7 at all ambient pH values. Hemolymph pH
of A. aegypti is influenced by ambient pH (P<0.005;
single factor ANOVA), decreasing by approximately 0.1 pH units to pH 7.6 in
larvae reared in pH 7 or 4 (Fig.
1). Hemolymph pH of A. aegypti is thus more acidic than
that of O. taeniorhynchus at pH 4 and 7 (pH 4: P<0.05, pH
7: P<0.002, two-tailed t-test, Microsoft Excel).
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Effects of pH on growth and development of A. aegypti and O. taeniorhynchus
When grown in water containing buffered 3.5 g l1 NaCl,
larval stage duration and growth rate (wet mass) but not pupal wet mass are
differentially affected by pH in the two species
(Fig. 2, Table 2). Nevertheless, for all
three life history parameters, the influence of pH, even when statistically
significant, is small in magnitude when compared with the much larger
influences of species and sex.
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Developmental time
Developmental time differs significantly between the two species, with
A. aegypti taking longer overall to pupate than O.
taeniorhynchus (Fig. 2A,
Table 2). However the
difference between species in development time depends significantly on both
pH and sex (indicated by the significant speciesxsexxpH
interaction; Table 2). A
separate model exploring this interaction detects significant differences
between the sexes in the effect of rearing pH in O. taeniorhynchus
(significant sexxpH interaction,
Table
3,Fig. 2A). Further
submodels run for each sex show that developmental time of female O.
taeniorhynchus is significantly lower at intermediate pH (4 vs.
7, P<0.05; 7 vs. 11, P<0.005) whereas male
developmental time does not fluctuate much across a wide pH range
(Fig. 2A; P>0.05
for effect of pH). Averaged across both sexes, the overall effect of pH on
development time is not statistically significant
(Table 3). In A.
aegypti, both males and females pupate significantly earlier at pH 7 than
in pH 4 or in pH 11, but do not differ in development time in pH 4 and 11
(P>0.001, P<0.005, P=0.59, respectively for
contrasts within the one-species model). Under the conditions of this study,
males and females of this species do not differ from one another overall in
their development times nor do they differ in their response to pH
(Fig. 2A,
Table 3).
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Pupal wet mass
Pupal wet mass differs significantly between A. aegypti and O.
taeniorhynchus (Fig. 2B,
Table 2). The full model
including data on both species detects a marginally significant maximum wet
mass at neutral pH, with lower mass at the pH extremes
(Fig. 2B, Table 2). The models run
separately for each species detect significant differences in pupal mass
across pH values in O. taeniorhynchus but not in A. aegypti
(Table 3). However, even for
O. taeniorhynchus the effect of pH is small (maximum difference among
pH values are: males; 9%, females 5%). In both species, females are
substantially larger at pupation than males
(Fig. 2B), regardless of
rearing pH. This significant effect is detected in all three models (Tables
2,
3).
Growth rate of wet mass
The growth rate of wet mass is higher for O. taeniorhynchus than
for A. aegypti, and higher in females than in males in both species
(Fig. 2C, Tables
2,
3). Across species and sexes,
this rate differs significantly across the pH range, demonstrating a maximum
at pH 7 (Fig. 2C,
Table 2), but this effect of pH
depends marginally significantly both on the larva's sex and the species
(speciesxsexxpH interaction;
Table 2). Separate models run
for each species further investigating this interaction detect the overall
effect of sex and pH in both species, further supporting the results of the
single-species model, but fail to detect any significant interdependence
(Table 3).
Acclimation of A. aegypti larvae to extreme pH
Transfer experiments using larval A. aegypti demonstrate the
capacity for acclimatory responses to extreme pH in mosquito larvae.
Acclimation of larval A. aegypti to extreme pH increases the
tolerable range of pH, rather than shifting it
(Fig. 3A,B). Larvae always die
rapidly when placed directly into water of pH 3 or 12 on the day following
hatching (see Table 1). Larvae
acclimated to pH 4 or 11 for several days survive significantly longer upon
transfer to pH 3 or 12, respectively, than do controls reared in pH 7 before
transfer (Fig. 3A;
P<0.05, t-tests). Acclimation also allows survival of a
few larvae to pupation at each pH extreme. One of 27 larvae transferred to pH
12 after acclimation in pH 11 survived to pupation, as did 2 of 32 transferred
to pH 3 following acclimation in pH 4. In contrast, larvae reared for 3 days
in pH 7 always die rapidly upon transfer to either pH 3 or 12
[Fig. 3A, N(pH 3)=24,
N(pH 12)=10], demonstrating that it is acclimation and not larval age
that extends life at these extreme pH values.
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Larvae reared initially in pH 4 for 3 days, then transferred to pH 11, showed no decrease in the rate of successful pupation compared to larvae maintained in pH 4 (Fig. 3B; P>0.05, t-test). Similarly, larvae acclimated to pH 11, then transferred to pH 4, showed the same proportion of successful pupation as those maintained in pH 11 (Fig. 3B; P>0.05, t-test). Among males and females transferred from pH 11 to 4 and pH 4 to 11 following 3 days of acclimation, there were no significant differences in duration of the larval stage, pupal mass or growth rates when compared with controls, with the exception of reduced growth rates of females transferred from pH 11 to 4 (Fig. 3C, P<0.05, t-test). The reduced growth rates of females transferred from pH 11 to 4 were due to a combination of a small but non-significant increase in larval stage duration and a similar non-significant decrease in pupal mass (data not shown). These two non-significant effects summed to produce a significant decrease in growth rates by female larvae transferred from pH 11 to 4 (Fig. 3C, P<0.05; t-test). No such decrease was observed in males.
Differences in responses of species and sexes to pH
Despite the overall similarities in growth and developmental parameters
between species and sexes, some differences are observed. The duration of the
larval stage of O. taeniorhynchus is influenced by sex and not pH,
whereas larval stage duration of A. aegypti is influenced by pH and
not sex. Wet mass of O. taeniorhynchus is influenced by both sex and
pH, whereas only sex influences wet mass of A. aegypti. In several
instances we also detect evidence for differences between sexes within a
species. Developmental time of male O. taeniorhynchus is more robust
to changes in pH than is that of females. Growth rates are reduced by transfer
of female but not male A. aegypti from pH 11 to pH 4.
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Discussion |
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Prior reports have documented the remarkable abilities of larval mosquitoes
to tolerate waters characterized by differences in H+
concentrations of many orders of magnitude
(Keilin, 1932;
Kurihara, 1959
;
MacGregor, 1921
; Peterson and
Chapman, 1970). We extend those findings by demonstrating similar, broad pH
tolerances in species with very different salinity tolerances. The distinct
salinity tolerances of larval Aedes aegypti and O.
taeniorhynchus are due to differences in their rectal structure and
function. Freshwater species, including A. aegypti, have a rectum
composed of a single segment that acts to recover ions from the rectal
contents. The rectum of O. taeniorhynchus and other euryhaline
osmoregulators consists of two segments, with an anterior segment that
functions like the rectum of freshwater forms, and a salt-secreting posterior
segment that allows these species to survive in saline water (Bradley and
Phillips, 1975
,
1977
). Identical pH tolerances
in related freshwater and euryhaline species demonstrate that the salt gland,
found only in euryhaline osmoregulators, is not involved in pH regulation.
Survival of larvae of the mosquito Aedes dorsalis in highly alkaline
lakes has been attributed to Cl/HCO3 exchange
occurring in the anterior rectal segment
(Bradley and Phillips, 1977
;
Strange et al., 1982
,
1984
;
Strange and Phillips, 1985
).
The data presented here are consistent with these observations, and
demonstrate that pH regulation in acidic waters does not require a posterior
rectal salt gland either. The organs involved in pH regulation in acidic
waters have yet to be determined.
We were surprised to find that the pH tolerances of Aedes aegypti
appear to be similar in nominally salt-free water and in water containing 3.5
g l1 NaCl or sea salt. In aquatic animals, movement of
acid/base equivalents is generally coupled with movements of strong ions such
as Na+ and Cl
(Cooper, 1994;
Truchot, 1987
). In aquatic
insects in general, failure to survive in acidic water appears to be due to
failure to regulate Na+ rather than failure to regulate hemolymph
pH. This is presumably due to sacrifice of Na+ to maintain
hemolymph pH through Na+/H+ exchange
(Wiederholm, 1984
;
Havas, 1981
). In mosquitoes,
ion exchange mechanisms have been investigated primarily in terms of
Na+ and Cl uptake
(Stobbart, 1971
; Strange et
al., 1982
,
1984
;
Strange and Phillips, 1985
;
Patrick et al.,
2002a
,b
).
However, based on the coupling of Na+/H+ and of
Cl/HCO3 transport, one might
expect the range of tolerable pH to be greater in the presence of NaCl; no
such influence of NaCl was detected.
There are no major trade-offs between pH regulation in acidic and alkaline
conditions in these larvae. We find that acclimation to low and high limits of
the pH range allows larvae to tolerate even more extreme pH levels with no
loss of ability to survive at the other pH extreme. The ability to tolerate
sudden changes in pH from one extreme to the other suggests that major
qualitative rearrangements of transporter expression are not necessary when
faced with either a highly acidic or alkaline environment, while acclimatory
expansion of the pH range suggests that qualitative changes in capacity are
possible. We therefore hypothesize that the mechanisms of acid and base
excretion are physically separate and independent, as in the acid- and
base-secreting cells of the mammalian kidney
(Brown et al., 1992). The
ability to cope with rapid changes in pH may be a consequence of having
separate mechanisms for acid and base secretion, rather than an adaptation
providing the capacity to tolerate sudden changes in pH. However, these data
also suggest that there exist mechanisms that allow rapid adjustments of the
activity of existing transporters upon changes in ambient pH.
Extremes of ambient pH have little effect on growth and development of
larval mosquitoes, whereas species and sex exert strong influences. These
species thus exhibit life history parameters that are remarkably robust in the
face of an enormous range of ambient pH. The minor influence of pH on growth
and development is rather surprising, especially considering that the effects
of salinity on pupal mass and growth rates are much more dramatic
(Clark et al., 2004). The minor
influences of pH on pupal mass and growth suggest that either pH regulation is
not metabolically expensive at extreme pH or that larvae can compensate by
increasing dietary consumption and/or utilization of nutrients. One
physiological attribute that may contribute to their pH tolerance is their
method of gas exchange. Havens
(1993
) found that the three
most acid-sensitive species of aquatic insects were those with the greatest
surface area of external permeable structures. As air breathers, larval
mosquitoes do not require intimate contact between highly permeable
respiratory surfaces and the surrounding water.
Values obtained in the present study for hemolymph pH of O.
taeniorhynchus are similar to those obtained by Giblin and Platzer
(1984) in this species
(7.62±0.14), and those obtained by Strange et al.
(1982
) for hemolymph of
Aedes dorsalis reared at pH 10.5 under varying
HCO3 concentrations. The data presented here
extend these observations by demonstrating that both freshwater and euryhaline
mosquitoes can maintain these hemolymph pH values across a wide range of
ambient pH values (from 4 to 11 in these species). This remarkable pH
regulatory ability is the key to the ability of mosquito larvae to tolerate
such broad ranges of ambient pH. Ochlerotatus taeniorhynchus appears
to be a somewhat more effective regulator than A. aegypti, as
hemolymph pH of O. taeniorhynchus is constant across the entire pH
range whereas that of A. aegypti changes by 0.1 pH unit as ambient pH
increases from 7 to 11. Ochlerotatus taeniorhynchus thus has a
hemolymph pH that is higher than that of A. aegypti at low and
neutral pH. This change in hemolymph pH of A. aegypti with ambient pH
is not associated with any noticeable difference in survival rates or changes
in larval growth or development parameters, suggesting that it is not of great
physiological significance.
In conclusion, this initial study into the physiology of pH tolerance in larval mosquitoes describes what must surely be one of the most pH-insensitive groups of animals in existence. Not only do larvae survive across seven orders of magnitude of ambient H+ concentration, but instantaneous transfer from one extreme of the tolerable range to the other has little discernable effect on growth and development. Mosquito larvae are also unusual in that their acid tolerances in low Na+ water are similar to those in 3.5 g l1 NaCl.
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Acknowledgments |
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References |
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