Low temperature acclimated populations of the grain aphid Sitobion avenae retain ability to rapidly cold harden with enhanced fitness
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* Author for correspondence (e-mail: sjp822{at}bham.ac.uk)
Accepted 18 April 2005
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Summary |
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Key words: rapid cold-hardening, seasonal acclimation, aphid, Sitobion avenae, fitness
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Introduction |
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Although summer-acclimated insects may have a greater potential to
demonstrate a RCH response, in the Northern Hemisphere, the ecological
importance of this ability is more associated with low-temperature survival,
either in autumn and spring, when seasonal acclimation is incomplete or
receding, or during periods with rapidly fluctuating temperatures
(Lee et al., 1987;
Coulson and Bale, 1990
;
Kelty and Lee, 1999
). In other
words, in natural environments, the conditions under which rapid
cold-hardening might be induced are likely to be part of a trend of
decreasing, or generally low, temperatures that would have already triggered
the start of seasonal cold acclimation. As far as we are aware, this is the
first time that the ability to rapidly cold-harden has been investigated in
insects that have already undergone long-term, low temperature
acclimation.
The grain aphid Sitobion avenae Fabricus is a monoecious species
overwintering on grasses and cereals as holocyclic (eggs) and anholocyclic
(aphids) clones. The anholocyclic clones are chill susceptible
(Bale, 1996), with high levels
of pre-freeze mortality after brief exposures to relatively high sub-zero
temperatures (Knight and Bale,
1986
). A recent study (Powell
and Bale, 2004
) found that laboratory populations of an
anholocyclic clone of S. avenae reared at 20°C are able to
rapidly cold-harden in response to 23 h acclimation at 0°C, or by
slow cooling (0.1°C min-1) from 10°C. It was hypothesised
that rapid cold-hardening may be relatively more important in insects such as
aphids (compared with longer-lived species) because their short generation
times, even in winter, may prevent the `full development' of seasonally
induced cold-hardiness. Whilst it is known that aphids reared at 10°C for
one or more generations are more cold hardy than those maintained continuously
at 20°C (Clough et al.,
1990
), the ability of such low-temperature acclimated aphids to
also rapidly cold-harden has never been investigated.
This paper describes a series of experiments that investigated the ability of populations of S. avenae, acclimated at 10°C, to further increase their cold tolerance via exposure to temperature regimes known to induce rapid cold-hardening in aphids reared at 20°C, and assesses the ecological costs (development, longevity and fecundity) associated with this response.
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Materials and methods |
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Determination of discriminating temperature
The discriminating temperature, defined as the exposure temperature
resulting in approximately 20% survival, was determined by directly
transferring aphids from the culture temperature (10°C), to progressively
lower sub-zero temperatures for 3 h, before re-warming at 1°C
min-1 to 10°C. Ten replicates of 10 aphids were placed inside
plastic Eppendorf tubes, which were then placed inside glass boiling tubes
stoppered with cotton wool and lowered into a Haake alcohol bath (F8-C50) set
to the desired sub-zero temperature. A thermocouple placed inside an Eppendorf
tube measured the temperature experienced by the aphids. Following the
low-temperature treatment, aphids were placed on a recovery tray (strips of
barley leaf on moist tissue under the lid of a small Petri dish) and returned
to 10°C. Survival was assessed after 24 and 72 h. Surviving aphids were
defined as those capable of co-ordinated movement.
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Statistical analysis
All survival data was arcsine and square root transformed to ensure a
normal distribution, then analysed using analysis of variance (ANOVA) and
Tukey's multiple range test.
Ecological costs of rapid cold-hardening
The occurrence of possible deleterious effects associated with rapid
cold-hardening on the development, longevity and fecundity of first instar and
adult S. avenae was investigated by establishing three treatment
groups: (i) control: aphids maintained continuously at 10°C; (ii) rapidly
cold-hardened: aphids acclimated at 0°C for the periods of time producing
the highest survival at the discriminating temperature (2 h fornymphs and 30
min for adults) and then returned to 10°C; (iii) exposed: aphids rapidly
cold-hardened at 0°C for 2 h (nymphs) or 30 min (adults), and then exposed
to the appropriate discriminating temperature for 3 h before being returned to
10°C. For each treatment five groups of 10 aphids were placed inside
plastic Eppendorf tubes, which were then placed inside glass boiling tubes
stoppered with cotton wool and lowered into a Haake alcohol bath (F8-C50) set
to the desired temperature regime. Following treatment, aphids were placed
singly onto cut barley inside an Austin tube. Development and reproduction
were monitored daily by counting and removing moulted cuticles or new-born
nymphs respectively.
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Results |
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Detection of a rapid cold-hardening response: varying the acclimation period at 0°C
When aphids were transferred directly from the culture temperature
(10°C) to their respective discriminating temperature, 26% of first instar
nymphs and 22% of adults survived (Fig.
2). First instar nymphs required a minimum acclimation period of
30 min at 0°C to significantly increase survival from 26% to 62%, compared
with the `direct plunge' exposure at 11.5°C
(F8,32=19.1, P<0.01). Nymphal cold hardiness
reached its maximum following a 2 h acclimation period at 0°C with 96%
surviving 3 h at the discriminating temperature; acclimation for longer than 4
h resulted in a trend of steadily decreasing survival that became
significantly different from maximum survival after 8 h of acclimation
(Fig. 2).
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Detection of a rapid cold-hardening response: varying the rate of cooling
Fig. 3 shows mean percentage
survival of 10°C-reared first instar nymphs and newly moulted
pre-reproductive adults, cooled from 10° to 0°C at 1°, 0.1°
and 0.05°C min-1, prior to a 3 h exposure at the respective
discriminating temperatures. At all three rates of cooling, survival of first
instar nymphs after 3 h at the discriminating temperature, was significantly
increased compared with a direct plunge to 11.5°C for the same
period (F3,12=25.1, P<0.01). The fastest
cooling rate of 1°C min-1 increased survival from 25% to 78%;
maximum cold tolerance was induced by cooling at 0.1°C min-1,
resulting in 86% of nymphs surviving exposure at the discriminating
temperature. In contrast, the cold hardiness of adult S. avenae was
not significantly increased by any of the cooling rates investigated.
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Ecological cost of rapid-cold hardening: aphids treated at the first instar nymph stage
Data on the development, fecundity and longevity of the three treatment
groups (control, rapidly cold-hardened and exposed) are shown in
Table 1. Development time was
not significantly affected by the rapid cold-hardening or exposure treatments
compared with the control. Daily fecundity increased from the control through
rapid cold-hardening to the exposed treatments, suggesting that increasing the
level of cold stress increases fecundity in surviving aphids. Compared with
the control, rapid cold-hardening did not affect daily reproduction; however,
the exposed treatment resulted in a significant increase (1.6±0.1
nymphs per day) compared with both the control (1.1±0.1 nymphs per day)
and RCH treatments (1.3±0.1 nymphs per day)
(F2,73=13.3, P<0.01), fecundity (recorded over the
entire life span) followed a similar pattern with the exposed treatment
producing a significantly higher number of nymphs (50.7±2.6)
(F2,73=5.4, P<0.01) compared with both the RCH and
control treatments, which in turn were not significantly different. Mean
length of reproductive life and mean longevity were similar in all three
treatments.
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Ecological cost of rapid cold-hardening: aphids treated at the newly moulted pre-reproductive adult stage
Neither the RCH nor exposure treatments affected the daily fecundity of
aphids treated at the newly moulted pre-reproductive adult stage
(Table 1). However, as with
first instar nymphs, mean total fecundity increased significantly with
increasing levels of cold stress (F2,73=22.9,
P<0.01); RCH treatment increased mean total fecundity from
16.8±1.5 (control) to 33.1±1.7 nymphs, with a further increase
to 34.6±2.7 nymphs in the exposed treatment. The RCH and exposed
treatments significantly increased the length of reproductive life and
longevity compared with the control.
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Discussion |
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As far as we are aware, there have been no previous studies comparing rapid
cold-hardening between low-temperature acclimated and non-acclimated
populations of the same species. The first report of rapid cold-hardening in
an aphid was with an anholocyclic clone of S. avenae reared at
20°C (Powell and Bale,
2004). The data for populations of S. avenae reared at
20°C provide a comparative baseline by which to assess the occurrence and
extent of RCH in acclimated aphids. Thus at 20°C, survival of nymphs and
adults increased from 18% and 16% at their respective discriminating
temperatures (8°C and 8.5°C) to 83% and 68%. There were
no ecological costs (effects on development, longevity or reproduction)
associated with rapid cold-hardening of nymphs or adults, compared with aphids
maintained continuously at 20°C, but exposure of rapidly cold-hardened
aphids at their respective discriminating temperatures significantly reduced
fecundity and longevity of both age groups
(Powell and Bale, 2004
).
Recent research has found that aphids acclimated at 10°C for three
generations are more cold-hardy than populations maintained at 20°C, as
revealed by a lowering of the LTemp50 (temperature that results in
50% mortality of the experimental population) of nymphs from 8.0°C
to 16.0°C, and of adults from 9.3°C to 13.5°C
(S.J.P. and J.S.B., unpublished data). This study demonstrated that a greater
level of cold tolerance was also reflected in the rapid cold-hardening
response, in which the discriminating temperature was lowered from
8° to 11.5°C for nymphs and 8.5° to
12°C for adults. Importantly, these low-temperature acclimated
aphids still retained the ability to rapidly cold-harden, increasing survival
at the discriminating temperature from 26% to 96% and 22% to 70% for nymphs
and adults, respectively. In contrast to aphids reared at 20°C, which
demonstrated no ecological costs or benefits as a result of rapid
cold-hardening (Powell and Bale,
2004
), the fecundity of low-temperature acclimated aphids treated
at the adult stage, increased significantly after rapid cold-hardening at
0°C, and was further significantly increased (compared to the control)
after exposure at the discriminating temperature. Supporting the hypothesis
that RCH can re-set the thermal thresholds for certain behaviours such as
reproduction (Bale, 2002
). The
longevity of adults was also significantly increased after 30 min of
acclimation at 0°C. However, the fecundity and longevity of aphids treated
at the first instar nymph stage was unaffected by rapid cold-hardening at
0°C for 2 h. The absence of any deleterious effects of RCH on the
fecundity and longevity of S. avenae contrasts with the results of
Coulson and Bale (1992
) who
found that in Musca domestica, both of these performance parameters
were significantly decreased by RCH. However, Broufas and Koveos
(2001
) reported that the cost
of RCH on the post diapause reproductive output of Euseius (Amblyseius)
finandicus, was negligible, and a recent study by Shreve, Kelty and Lee
(Shreve et al., 2004
) reported
an increase in successful mating of Drosophila melanogaster following
RCH, compared with flies exposed directly to low temperature. It is therefore
clear that the ecological cost of RCH varies greatly between different
species. When the ability of low-temperature acclimated nymphs and adults to
rapidly cold-harden are compared, the main difference between the two life
stages is the increase in nymph survival at their discriminating temperature
after slow cooling from 10° to 0°C, but the absence of a similar
response in adults.
In a wider context, RCH may influence the overwintering success of
anholocyclic aphids, and hence their economic importance. Winter exerts a
major influence on the annual population dynamics of aphid species with
anholocyclic clones, particularly the timing of the spring migration and the
number of migrating aphids (Harrington et
al., 1989; Werker et al.,
1998
). It is known that aphids have low supercooling points
(<20°C) and die before they freeze (LTemp50 of
8.1°C for Myzus persicae reared at 20°C), but can
acclimate when reared at lower temperatures (LTemp50 of M.
persicae is lowered to 11.5°C after two generations at
10°C) (Clough et al.,
1990
). However, the overwintering biology of anholocyclic aphids
differs from that of most other insects in one key area: aphids continue to
develop and reproduce throughout winter. As a consequence, an aphid found in
the field in early spring is likely to be the second, third or fourth
generation descendant of the individual that entered the winter 45
months earlier. Owing to these short generation times, it has been suggested
that aphids may have a comparatively limited ability to seasonally acclimate,
and that rapid cold-hardening may be relatively more important in these
insects (Powell and Bale,
2004
). It is certainly the case that aphid mortality increases
rapidly over a narrow range of temperatures (5°C to
15°C; Knight and Bale,
1986
), hence even modest decreases in the lethal temperature may
be important for the winter survival of anholocyclic clones.
In summary, this study demonstrates that low-temperature acclimated S. avenae retain the ability to rapidly cold-harden as a result of a short acclimation period at 0°C (nymphs and adults) or by slow cooling at rates between 1 and 0.05 min-1 (nymphs only). This suggests that rapid cold-hardening may be an important component in the overwintering survival of S. avenae and other aphids with anholocyclic clones, with the additional benefit of increasing longevity and fecundity.
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Acknowledgments |
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