Differences in the effects of salinity on larval growth and developmental programs of a freshwater and a euryhaline mosquito species (Insecta: Diptera, Culicidae)
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, salinity, life history, growth rate, developmental rate, Aedes aegypti, Ochlerotatus taeniorhynchus, insect
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Introduction |
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While survival to adulthood has obvious fitness consequences, environmental
influences on more subtle aspects of growth and development are also important
in determining fitness. Mosquito larvae face a number of threats, including
predation and desiccation of habitats, and mosquitoes are typically
characterized by rapid completion of the life cycle. Rapid development allows
multivoltine insects, including mosquitoes, to complete more generations
during the breeding season and accomplish more explosive population growths
during favorable periods. Among insects, females are generally larger than
males, and female size is related to fecundity. There thus exists a selective
trade-off between rapid development and size at maturity. Phenotypic traits
such as growth rates and mass are affected by geneenvironment
interactions, and a number of environmental factors are known to influence
mosquito growth and development. These include salinity, temperature, density,
food supplies, and physical size and shape of the larval habitat
(Clark et al., 2004; Nayar,
1968
,
1969
;
Nayar and Sauerman, 1970
;
Trpis and Horsfall, 1969
;
McGinnis and Brust, 1983
).
Because so many factors influence growth rates of larval mosquitoes, comparisons of the responses of different species to changes in a particular environmental parameter must be carefully controlled. To this end, in this study larvae of the two species were reared at the same density, in the same volume of water, and in identical containers. The same feeding protocol, photoperiod and temperature regimes were also used. These factors are all known to influence growth and development. In this way, the responses of larvae of the two species to salinity, when subjected to conditions within their respective physiological capacities, could be compared directly.
The salt-secreting gland found in euryhaline species such as O. taeniorhynchus, lacking in freshwater forms including A. aegypti, is hypothesized to alter the energetics of ionoregulation sufficiently to impact growth and developmental parameters. We predicted that A. aegypti and O. taeniorhynchus would respond in a similar way to increased salt concentrations at the low end of the salinity range, due to their shared mechanisms for survival in freshwater, and that their responses would diverge as salinity increased beyond that typically experienced by freshwater larvae. Instead, what we found was that salinity has fundamentally different effects on growth and developmental programs in the two species examined.
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Materials and methods |
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Solutions
Instant Ocean artificial seawater (Aquarium Systems, Mentor, OH, USA) was
used to make up rearing solutions. Larval A. aegypti were reared in
concentrations of 0, 3.5, 7, 10.5, 14 and 17.5 g l1
(encompassing the range from deionized water to approximately 50% seawater),
while larval O. taeniorhynchus were reared in concentrations of 0, 7,
14, 21, 28 and 35 g l1 (encompassing the range from
deionized water to approximately full-strength seawater). The osmotic
pressures of these sea salt solutions were determined to be as follows (g
l1: mOsm l1) using a Wescor 5500 Vapor
Pressure Osmometer (Logan, UT, USA): 0, OP not measured; 3.5, 83; 7, 167.5;
10.5, 278; 14, 356; 17.5, 442; 21, 528; 28, 695.6; 35, 897.
Statistical analyses
Effects of salinity were modeled using mixed linear models (SAS mixed
procedure, SAS Institute Inc. 1997). Full models that include species,
salinity, salinity squared, sex and all interactions were fitted in order to
compare salinity dependence between the two species. These were followed with
separate models fitted for each species, with sex, salinity, salinity squared
and their interactions. This quadratic term was included to fit curvature in
the relationship between life history parameters and salinity. For each of the
five life history parameters, duration of the larval stage, pupal wet mass,
pupal dry mass, growth rate of wet mass and growth rate of dry mass, a model
containing sex, salinity and salinity squared was first fitted. Where the
quadratic term was not statistically significant, it was dropped from the
model and a purely linear model was fitted. Because preliminary models found
no significant sex-dependent response to salinity, no such interaction terms
are included in the final best-fit models. A single life history parameter,
days to pupation, was log-transformed to improve normality.
Path diagrams of the relationships among salinity, growth parameters
(log-transformed days to pupation and assimilation of dry mass), and final
pupal dry mass were constructed using the SAS REG procedure with the STB
option (Pedhazur, 1982; SAS
Institute Inc. 1997). Separate diagrams were made for each sex and species,
but within each species the two sexes are shown in a single diagram, since no
substantial differences remained after standardizing for overall differences
in larval size.
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Results |
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Effects of species, sex and salinity on life-history variables
Aedes aegypti was reared at salinity intervals of 3.5 g
l1 whereas O. taeniorhynchus was reared at
intervals of 7 g l1. Two response variables were measured:
duration of the larval stage and pupal mass (wet and dry). From these
parameters the mean growth rates of wet and dry mass over the larval stage
were calculated. Mixed linear models run on a reduced data set including only
shared salinities show that the two species differ with respect to all five
life-history variables (main effect of species, P<0.01 for all
life history response variables). In addition, the dependence of these
variables on sex and salinity is significantly affected by species
[sexxspecies, P<0.01 for mass and wet growth rate;
salinitxspecies, P<0.0001 for ln(days to pupation); salinity
squaredxspecies, P<0.005 for wet and dry growth rate and
log-transformed days to pupation]. Separate models were therefore used to
describe each species (Table
1).
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Duration of the larval stage
Within a species, duration of the larval stage is sex-dependent
(Fig. 2; Table 1). Both sexes respond to
salinity in a similar way, but females consistently take longer to pupate than
do males. Distinct patterns of response to salinity are observed between
species. The duration of the larval stage of O. taeniorhynchus is
positively related to salinity across the entire range tested (035 g
l1) (Table
1). Changes in duration of the larval stage due to salinity are
less than 10% of the total. The pattern observed in larval A. aegypti
is quite different. Developmental rates of these larvae show a -shaped
curve, with most rapid development occurring at 7 g l1
salinity, and developmental time increasing abruptly as salinity increases
above that value (Fig. 2). This
is reflected in the significant effect of the quadratic term for salinity
(salinity squared) on log-transformed duration of larval stage in this species
(Table 1). Male developmental
duration increases by 24%, while female duration increases by 15%, as salinity
increases from 7 g l1 to 14 g l1
(Fig. 2). This is the same
range over which survival decreases precipitously
(Fig. 1).
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Pupal mass
Clear differences are observed between species and sexes in pupal mass.
Within each species females are larger than males
(Fig. 3A,B; Table 1). Ochlerotatus
taeniorhynchus is larger than A. aegypti. Salinity affects pupal
mass in both species studied. Pupal wet and dry masses of male and female
O. taeniorhynchus increase with salinity across the entire range of
salinities tested (Fig. 3A,B;
Table 1). As salinity increases
from 0 to 35 g l1, in O. taeniorhynchus male mass
increases by 18% (wet mass) and 26% (dry mass) while female mass increases by
17% (wet mass) and 25% (dry mass). The increase in wet mass in O.
taeniorhynchus across salinities shows significant curvature
(salinity2 term, Table
1), with mass remaining relatively constant across the midrange of
salinities and greater increases in mass over a given salinity increment at
higher salinities. In contrast, pupal mass (wet and dry) of A.
aegypti decreases linearly as salinity increases from 0 to 14 g
l1 (Fig.
3A,B; Table 1). Male wet mass decreases by 21%, while female wet mass decreases by 14%, across
this salinity range. The effects of salinity on the two species thus differ
both in sign and in curvature.
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Growth rate
Growth rates (rates of assimilation of wet and dry mass) are strongly
dependent on species and sex (Table
1, Fig. 4A,B). The
effects of salinity are species dependent. Growth rates (wet and dry mass) are
significantly greater in females than in males of each species
(Table 1). For O.
taeniorhynchus, dry mass growth rate increases significantly with
increasing salinity, but wet mass growth rate remains constant across
salinities reflecting a decrease in percent body water with salinity
(P<0.001, mixed linear model including sex and salinity). In
A. aegypti, growth rates of wet and dry mass both decrease
significantly with salinity and this decrease is accelerated at the greatest
salinities (Table 1;
salinity2). Salinity has no significant effect on the percent body
water of A. aegypti (P=0.61, mixed linear model, including
sex and salinity).
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Differences in patterns of growth and development
The differences in the effects of salinity on growth and development in
these two closely related species are summarized in the path diagrams shown in
Fig. 5. Thedifference in sign
of the effect of salinity on growth rate in the two species explains much of
the final difference in the effects of salinity on pupal dry mass, a major
fitness component, since growth rate is strongly positively correlated with
mass at pupation. In O. taeniorhynchus pupal dry mass is highest at
high salinity (Fig. 3).
Multiplication of the path coefficients along each side of the diagram reveals
that 40% of this correlation can be explained by salinity's effects
via the time to pupation, while most of the remaining 60% of the
explained variability is due to its effects on dry growth rate. In contrast,
the net effect of increasing salinity for A. aegypti is a decrease in
pupal dry mass (Fig. 3), mainly
through its effect of decreasing growth rate, as 70% of the explained
variability in dry mass at pupation is via the negative effects of
salinity on assimilation of dry mass. The increased larval duration that
occurs at high salinity explains close to 30% of the variability in pupal dry
mass, but does not fully compensate for the negative effect of salinity on dry
growth rate.
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Discussion |
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The different physiological mechanisms used by these two species to deal with ionic loads are expected to lead to differences in the effects of salinity on the balance between nutrient assimilation and nutrient expenditure during development. This is expected to lead in turn to differences in responses of growth and developmental parameters to salinity in the two species. We originally hypothesized that the two species would react in a similar way at low salinities, and differences would emerge and increase in magnitude as the salinity departed from values normally experienced by freshwater larvae. What we found instead is that the developmental programs of the two species respond to salinity in fundamentally different ways. The effects of salinity on growth and development of O. taeniorhynchus are due to positive direct influences of salinity on assimilation of dry mass, and on larval stage duration, both of which positively influence mass at pupation (Fig. 5). Although the increase in larval stage duration in response to increased salinity is actually larger than the increase in dry growth rate (Figs 2, 4), because the latter so strongly influences pupal mass, it is via its effect on growth rate that salinity most strongly influences final dry mass (Fig. 5). In O. taeniorhynchus, pupal mass is a function of larval stage duration, which is positively related to salinity (see Figs 3, 5). Thus, it appears that in O. taeniorhynchus the decision to pupate is uncoupled from information about pupal mass, so that increased larval stage duration and growth rate results in greater mass as salinity increases. In A. aegypti, a very different pattern emerges. Salinity influences mass through a curvilinear effect on larval duration, which lengthens the time to pupation at high salinities (Table 1, Figs 2, 5). However increased salinity also decreases larval growth rate in A. aegypti (Table 1, Figs 4, 5). As a result, despite the positive correlation between increased larval duration and mass at pupation, the increase in this trait at high salinity cannot fully compensate for negative effects of salinity via growth rate, resulting in a net negative effect of salinity on pupal dry mass (Table 1, Figs 2, 5). As salinity increases above 7 g l1, A. aegypti may partially compensate for salinity-induced changes in growth rates by adjusting developmental time, delaying pupation until the animal has acquired the critical mass for pupation and thereby maintaining pupal mass. The overall negative slope of this relationship shows that larvae in more saline water delay pupation but still pupate before they attain the mass that they would have attained in less saline conditions. This pattern suggests that A. aegypti assesses both larval stage duration and mass, reaching a compromise between rapid completion of the larval stage and maintaining ideal mass. Salinity thus influences developmental programs in fundamentally different ways in the two species investigated.
The increment in salinity that most dramatically delays development of A. aegypti is that over which the hemolymph osmotic pressure approaches the osmotic pressure of the environment. In larval mosquitoes, feeding leads to ingestion of the medium and may thus contribute to ionic loads under saline conditions. The decrease in growth rate of A. aegypti at greater salinities (Table 1, Fig. 4) may be due to decreased feeding rates to avoid ingestion of ions at greater rates than can be eliminated by the excretory system, and/or by decreased assimilation of nutrient stores due to increased metabolic demands of osmo- and iono-regulation at elevated salinities. This pattern could be explained by relatively slow growth rates at the time that they reach the critical mass for pupation, so that pupation occurs before significant additional mass has been accumulated. These data also suggest that a significant portion of the energy budget of larval A. aegypti is used for ionoregulation at higher salinities within the tolerable range.
In situations where the commitment to pupate occurs at a given mass, larger
size together with delayed development, as occurs in O.
taeniorhynchus (Table 1,
Fig. 2), should occur if
environmental conditions delay early growth until physiological adjustments
have been made. If growth is especially rapid following these adjustments,
then given fixed times between reaching critical mass and pupation, the mass
of the insect would overshoot the target. Intriguingly, these data resemble
those describing the relationship between adult size and developmental
temperatures, in which larvae develop more slowly at lower temperatures but
reach greater size (Clements,
2000).
The trade-off that exists between completing development quickly and attaining large size appears to lead to selection for increased growth rates in O. taeniorhynchus relative to A. aegypti, and in females of both species relative to males. It is tempting to speculate that the differences between the growth rates of A. aegypti and O. taeniorhynchus (Figs 2, 3, 4) are evolved traits driven by selective pressures. This is supported by the observation that the differences in growth rates of the two species are paralleled by differences in the behavior of their larvae and pupae. Larvae and pupae of O. taeniorhynchus swim quite rapidly, darting around the container in rapid bursts. A. aegypti larvae on the other hand swim slowly, and have a much greater tendency to mass together in the darkest corner of the rearing dish (T. M. Clark, unpublished observation). Ochlerotatus taeniorhynchus also completes the pupal stage within 48 h at 26°C, whereas the pupal stage of A. aegypti typically lasts more than 48 h at this temperature (T. M. Clark, unpublished observation). The reason for greater pupal mass of O. taeniorhynchus is likely to involve selective pressures acting on adults, such as selection for increased fecundity, possibly driven by increased mortality of larvae in their less-protected larval habitats. Similarly, the differences in growth rates and swimming speeds are likely to be driven by different selective pressures experienced by larvae of the two species in their natural habitats. Ochlerotatus taeniorhynchus larvae live in salt marshes, where predators such as fishes are common and rapid evaporation of temporary pools occurs. Larval A. aegypti, on the other hand, live in very small bodies of water such as tin cans and discarded tires, habitats unlikely to contain such predators. The difference in growth rates between the species increases with increasing salinity reflecting their different mechanisms of iono- and osmo-regulation.
A large number of environmental parameters influence growth and development
in larval mosquitoes. These sources of variability may explain some of the
discrepancies between the results of the current study and those of Nayar
(1969), who observed that size,
dry mass and percent lipid of O. taeniorhynchus decrease with
increasing salinity. It is possible, but unlikely, that the differences are
due to evolution of the laboratory strain used in the two studies during the
30+ years since the studies of Nayar
(1969
). The differences between
the results of that study and the present one more probably result from
different rearing conditions, such as feeding regimens. Similarly, the
significance of comparisons between the present work and the work of McGinnis
and Brust (1983
) is not clear.
In their study, the euryhaline Aedes togoi showed a
-shaped
response of developmental time to salinity, similar to the response of A.
aegypti. However, unlike both A. aegypti and O.
taeniorhynchus (this study), A. togoi showed the slowest
development in the most dilute water. It is possible that this species
exhibits yet a third pattern of response although once again we suspect that
environmental conditions contribute to the observed differences.
This is the first study to directly compare growth and development of two
species under the same environmental conditions in order to avoid such
artifacts. Patrick et al.
(2002a,b
)
have documented surprisingly diverse mechanisms of ionoregulation among
freshwater species, and even among populations within a species. The present
study shows that fundamental differences in mechanisms by which growth and
development respond to environmental influences can also occur among closely
related species.
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Acknowledgments |
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References |
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