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First published online November 17, 2006
Journal of Experimental Biology 209, 4690-4700 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02563
Photoperiod-induced plasticity of thermosensitivity and acquired thermotolerance in Locusta migratoria
Department of Biology, Queen's University, Biosciences Complex, Kingston, ON, K7L 3N6, Canada
* Author for correspondence (e-mail: rodgersc{at}biology.queensu.ca)
Accepted 27 September 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: central pattern generator, heat shock, insect, life history, locust, Locusta migratoria, photoperiod, thermosensitivity, thermotolerance, ventilation
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Therefore,
survival depends on the ability of neural circuits to adapt to temperature
fluctuations, allowing the animal to carry out important behaviours properly.
It is well established that prior exposure to a high but sub-lethal
temperature stress (heat shock: HS) leads to a state of improved
thermotolerance in which the animal can continue to generate motor patterns
and thus behave normally under previously non-permissive conditions
(Robertson et al., 1996;
Barclay and Robertson, 2000;
Newman et al., 2003;
Robertson, 2004a;
Robertson, 2004b;
Money et al., 2005). The
mechanisms mediating acquired thermotolerance have not been fully elucidated;
however, it is conceivable that the short- and long-term phenotypic changes
induced by a prior stress are influenced by environmental variables such as
photoperiod.
In poikilothermic animals, continued behaviour at normally lethal
temperatures indicates improved performance of the nervous system. Development
of thermotolerance is especially important for poikilothermic animals that
lack the physiological mechanisms for maintaining a stable body temperature,
and more so for those poikilotherms that are exposed to large fluctuations in
ambient temperature on a regular basis. The locust Locusta migratoria
is poikilothermic and native to sub-Saharan Africa. In their desert ecology,
L. migratoria are routinely exposed to extremes in heat that can
surpass 40°C, and internal temperature can be as much as 10-12°C above
ambient during vigorous activities such as flight
(Weis-Fogh, 1956). In order to
cope in this environment, it is likely that the locust has evolved
physiologically relevant ways of protecting neural function from heat stress.
Thus, the locust is an excellent model for studies of thermosensitivity,
thermotolerance and the HS response. Moreover, its relatively simple nervous
system provides a unique opportunity for examination at the cellular level in
live and semi-intact preparations.
One mechanism believed to underlie induced thermoprotection of neural
circuits is enhanced expression of heat shock proteins (Hsps). Locusta
migratoria adults exposed to 45°C for 0.5-4.5 h experience coincident
enhanced Hsp expression and improved thermotolerance during a subsequent,
normally lethal temperature stress (Whyard
et al., 1986). A twofold increase in expression of heat shock
protein 70 (Hsp70) was found in locusts after being exposed to HS
(Qin et al., 2003). Hsps have
a number of roles in the cell, including protein chaperoning
(Feder and Hofmann, 1999) and
cytoskeletal stabilization (Feder,
1996; Liang and MacRae,
1997), and it has been suggested that Hsps may have a direct or
indirect role in downregulation of K+ currents as seen in HS
locusts (Ramirez et al.,
1999). Induction of Hsps and other aspects believed to be involved
in thermotolerance are metabolically expensive and acquired thermotolerance is
thus subject to energetic constraints. In an effort to minimize costs for
animals that are exposed to widely fluctuating temperatures, it is a
reasonable supposition that the HS response is plastic.
The benefits of having a plastic HS response are many. For example,
plasticity allows an organism to modify the strength of its HS response in
order to maximize protection against thermal damage while minimizing the
metabolic costs incurred (Parsons,
2003). Evidence that the HS response is plastic has been
demonstrated in intertidal animals that experience huge fluctuations in daily
temperature as a consequence of where they live
(Buckley et al., 2001;
Halpin et al., 2004). For
example, natural levels of Hsp72 and the threshold induction temperature of
this protein were found to be greater in mussels (Mytilus
californianus) inhabiting higher rocky intertidal areas, compared to
mussels of the same species inhabiting the lower edges of the vertical
distribution (Halpin et al.,
2004). This study provides evidence that variation in physical
conditions of closely spaced microhabitats can result in different natural
levels of protein damage and a different HS response, depending on thermal
history. Other studies have investigated the relationship between acclimation
temperatures and natural levels of thermotolerance
(Willhite and Cupp, Jr, 1982;
Moseley, 1997). Our goal was
to determine if daylength modulates the strength of the HS-mediated protection
in the locust.
Photoperiod is a significant environmental variable that has been shown to
affect cold tolerance (Kim and Song,
2000), heat stress resistance
(Sorensen and Loeschcke,
2004), and Hsp70 level
(Sorensen and Loeschcke, 2004)
of insects. We used photoperiod to probe and challenge a crucial neural
circuit in the locust to determine how the HS response is affected. A 12 h:12
h light:dark (L:D) photoperiod was considered the natural state and a longer
daylength photoperiod of 16 h:8 h light:dark (L:D) was chosen arbitrarily as a
more stressful daylength. We first measured phenotypic traits and lifespan to
determine long-term effects of daylength, then we compared the ability of the
nervous system to withstand heat stress in locusts reared under each
photoperiod. We used ventilatory motor pattern generation as a model system to
investigate plasticity of thermosensitivity and acquired thermotolerance in
response to variation in photoperiod.
Ventilation is a crucial motor activity to locusts and is an appropriate
neural circuit for investigation of the effects of photoperiod on acquired
thermotolerance. Ventilation is under the control of a central pattern
generator (CPG) located in the metathoracic ganglion (MTG)
(Hustert, 1975;
Bustami and Hustert, 2000).
Ventilatory networks are sensitive to many different kinds of stress,
including changes in internal concentration of gases such as CO2
(Gulinson and Harrison, 1996;
Henderson et al., 1998) and
changes in pH (Snyder et al.,
1980), as well as temperature changes
(Banks et al., 1975;
Lighton and Lovegrove, 1990;
Henderson et al., 1998;
Newman et al., 2003;
Tryba and Ramirez, 2003).
Locusts ventilate discontinuously when relaxed, but employ continuous
ventilation when stressed, which makes it easy to determine when motor pattern
generation has failed and recovered. The frequency of ventilatory bursts
increases with increased heat. This is believed to be an adaptive advantage
for the animal by increasing evaporative cooling, which in turn dissipates
heat and lowers internal body temperature
(Prange, 1990;
Prange, 1996).
Operation of the ventilatory CPG in locusts can be protected against high
temperature stress if subjected to a prior HS. Prior stress reduced whole cell
K+ currents (Ramirez et al.,
1999), which would result in prolonged action potentials and
reduced accumulation of extracellular potassium. Following a HS treatment of 3
h at 45°C, adult male locusts were capable of maintaining a ventilatory
rhythm significantly longer at sustained high temperatures than animals that
had not received this treatment (Newman et
al., 2003). Additionally, HS animals recovered more quickly
following hyperthermic failure, had a lower incidence of failure and a higher
incidence of recovery during subsequent stress.
We examined frequency of ventilatory bursts, thermosensitivity, and thermotolerance of the ventilatory motor pattern during increasing temperature stress and the effect of a prior HS on these parameters in locusts reared under 16:8 and 12:12 L:D regimes. We defined thermosensitivity as the immediate response to changing temperature, and thermotolerance as the ability to cope with high temperature stress, i.e. time-to-failure and time-to-recovery of the motor pattern in response to increasing and constant high temperature stress. We hypothesized that animals from different photoperiods would be uniquely thermosensitive, and that thermotolerance of 16:8 and 12:12 HS animals would be different.
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Materials and methods |
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Lifespan and general morphology
Lifespan was monitored for male and female L. migratoria raised in
16:8 and 12:12 photoperiods to determine if the difference in daylength led to
a difference in this fundamental life history trait. Animals were checked
daily and dead ones were counted and removed. At 2-3 weeks of adult age, a
total of 24 males and 24 females were removed from these cages, and basic body
measurements were taken. These animals were not returned to the colony. Body
measurements included mass, posterior femur length and head capsule width.
Femur length (F) and head capsule width (C) were measured
using Vernier calipers with 0.1 mm precision to generate the
F/C ratio, generally regarded as the most reliable
diagnostic indicator of phase in locusts
(Dirsh, 1951;
Dirsh, 1953). At 54 days of
adult age, the lifespan experiment was terminated, and survivors were counted.
Raw data were transformed to generate a Kaplan-Meier survival curve of
cumulative survival probabilities (SigmaPlot 8.0, SPSS Inc., Chicago, IL, USA)
Starting sample sizes were: 12:12 males, N=47; 12:12 females,
N=61; 16:8 males, N=57; 16:8 females, N=57.
Experimental treatments
Male and female locusts, approximately 1-4 weeks of adult age, were
randomly chosen from the 16:8 and 12:12 colonies and subjected to either a
control or HS pre-treatment. Locusts that received HS pre-treatment were
placed in a 2 liter ventilated plastic container in an incubator (45°C)
for 3 h. A beaker of water was also placed in the incubator to maintain high
humidity in order to prevent desiccation and minimize evaporative cooling of
animals during pre-treatment. Control animals were placed in a similar
container and left for 3 h at room temperature (21±2°C). All
animals were allowed 1-5 h of recovery following pre-treatment and all
experiments were performed between 13:00 h and 18:00 h to avoid potential
time-of-day effects. Experiments on animals receiving different pre-treatments
were interspersed over time with each other.
Animals were divided into eight experimental groups that allowed examination of the response of the ventilatory CPG to thermal stress depending on prior exposure, the sex of the animal, and photoperiod: (1) 12:12 control males, N=8; (2) 12:12 control females, N=8; (3) 16:8 control males, N=7; (4) 16:8 control females, N=12; (95) 12:12 HS males, N=9; (6) 12:12 HS females, N=8; (7) 16:8 HS males, N=8; (8) 16:8 HS females, N=9.
Experimental set-up
Following removal of legs, wings and pronotum, ventilatory muscle 161 was
exposed by making a dorsal midline incision and pinning the locust open onto a
corkboard, dorsal side up. The gut, air sacs and fat bodies were removed.
A Peri-Star peristaltic pump (World Precision Instruments Inc., Sarasota, FL, USA) was used for perfusion of standard locust saline into the body cavity which contained (in mmol l-1) 147 NaCl, 10 KCl, 4 CaCl2, 3 NaOH, and 10 Hepes buffer (pH 7.2) (all chemicals were from Sigma-Aldrich, Oakville, ON, Canada). The saline flow was directed onto the MTG where the ventilatory CPG is located and flowed through the animal toward the posterior end. Saline passed through a glass pipette wrapped in NichromeTM wire, and the temperature of the saline was controlled by varying the amount of current passed through the wire. Temperature at the ganglion was monitored using a thermocouple connected to a digital thermometer (BAT-12; Physitemp Instruments Inc., Clifton, NJ, USA).
An extracellular recording of the ventilatory motor pattern was obtained by placing a 0.1 mm diameter copper wire, insulated except at the tip, onto abdominal muscle 161. The recording was digitized using a DigiData 1200 Series Interface (Axon Instruments Inc., Union City, CA, USA) (e.g. Fig. 1) and displayed using Axoscope 9.0. The preparation was grounded by placing a silver wire in the posterior tip of the abdomen.
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Fig. 1 demonstrates that ventilation is a series of rhythmic electrical bursts in the abdominal muscles. Ventilatory frequency (mean ± s.e.m.) was calculated by averaging the reciprocal of the cycle period for all cycles of each animal during the first 20 min while the locust was allowed to acclimatize at room temperature and at every 5°C increase in temperature up to and including 45°C. Frequency at 40°C was used rather than frequency at 45°C in some of our analyses because there was a low incidence of failure of the motor pattern by the time the temperature ramp reached 40°C. Time-to-failure was measured as the time at failure minus the time at the beginning of the temperature ramp. Time-to-recovery was measured as the time at recovery minus the time at failure. Animals that did not fail before 30 min at 45°C or recover within 30 min at room temperature were not included in the time-to-failure and time-to-recovery analyses.
Statistical analyses
Data were plotted using SigmaPlot 8.0 using the mean as the measure of
central tendency and standard error (s.e.m.) to describe dispersion about the
mean. Two-way RM-ANOVAs, t-tests, z-tests, and Pearson
Product Moment Correlations were performed using SigmaStat 3.0 statistical
analysis software (SPSS Inc.). For the analysis of data shown in
Fig. 3 and
Fig. 4, we used JMP IN 5.1
statistical analysis software (SAS Institute Inc., Cary, NC, USA) to perform
ANOVAs with three subject factors (photoperiod, pre-treatment and the sex of
the animal). Post hoc Tukey tests were performed to determine which
groups drove the main effects. A 95% confidence interval was used to determine
significance among means.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Effect of photoperiod on longevity
We found significant differences in survival probability between groups
(Kaplan-Meier Survival Analysis, Gehan-Breslow test statistic=21.06,
P<0.001, d.f.=3) (Fig.
2C). Pairwise comparisons revealed that 12:12 females and males
had a significantly higher survival probability than 16:8 females and males,
respectively (Holm-Sidak pairwise comparisons: 12:12 females vs 16:8
females, t=15.2, P<0.01; 12:12 males vs 16:8
males, t=8.8, P<0.01). There were no sex differences in
survival probability within each photoperiod (Holm-Sidak pairwise comparisons:
12:12 females vs 12:12 males, t=0.33, P=0.57; 16:8
females vs 16:8 males, t=2.66, P=0.1).
Effect of photoperiod on temperature sensitivity of ventilatory motor pattern generation before and after HS
Frequency of ventilatory bursts increased as internal temperature was
increased in all groups (Fig.
3A,B). There was an effect of photoperiod on ventilatory frequency
during increasing temperature in control animals
(Fig. 3A). In addition, there
was an effect of photoperiod on thermosensitivity, i.e. the slope of the
relationship between ventilatory rate and temperature, in both control and HS
locusts (Fig. 3A,B). Frequency
of ventilatory bursts during a ramped increase in temperature was
significantly higher in 12:12 control locusts than in 16:8 control locusts
(two-way RM-ANOVA, P<0.001, F(1,59)=23.457)
(Fig. 3A). The ventilatory
rhythm of 16:8 and 12:12 control locusts also responded differently to
increasing temperature stress (significant interaction between temperature and
photoperiod: two-way RM-ANOVA, P<0.001,
F(5,59)=15.611) (Fig.
3A). There was no main effect of photoperiod on frequency of
ventilatory bursts during a ramped increase in temperature in HS locusts
(two-way RM-ANOVA, P=0.148, F(1,61)=2.309);
however, the ventilatory rhythm of 16:8 and 12:12 HS locusts responded
differently to increasing temperature stress (significant interaction between
temperature and photoperiod: two-way RM-ANOVA, P=0.025,
F(5,61)=2.785) (Fig.
3B).
16:8 and 12:12 locusts responded differently to constant high temperature stress before and after HS (Fig. 3C-F). 12:12 locusts ventilated significantly longer than 16:8 locusts when internal temperature was increased to and held at 45°C (16:8 control males and females vs 12:12 control males and females: t-test, t=-3.994, P<0.05, d.f.=29; 16:8 HS males and females vs 12:12 HS males and females: Mann-Whitney Rank Sum Test, t=266.000, P<0.05) (Fig. 3C,D). 12:12 locusts had a significantly shorter time-to-recovery of the ventilatory motor pattern following hyperthermic failure than 16:8 locusts (16:8 control males and females vs 12:12 control males and females: t-test, t=3.547, P<0.05, d.f.=24; 16:8 HS males and females vs 12:12 HS males and females: Mann-Whitney Rank Sum Test, t=120.000, P<0.05) (Fig. 3E,F).
Percentages of locusts that failed and recovered
Failure of the ventilatory motor pattern occurred during the temperature
ramp before 45°C in some instances, and some animals continued to generate
a rhythm for 30 min at 45°C. The percentage of locusts whose motor pattern
failed before 40°C, 45°C and after 30 min at 45°C as well as the
percentage of locusts whose ventilatory rhythm recovered before 30 min at RT
following hyperthermic failure are significant measures of thermotolerance and
are presented for each group in Table
1.
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There was a small percentage of locusts whose motor pattern failed before 40°C in four out of the eight groups (Table 1). Overall, a higher percentage of all 16:8 locusts (5/36=14%) failed before 40°C compared to all 12:12 locusts (2/33=6%); however, there were no significant differences among groups (z-tests, P>0.05).
The total percentage of locusts whose ventilatory rhythm failed before the temperature ramp was complete increased from 40 to 45°C (Table 1). Overall, a higher percentage of all 16:8 locusts (12/36=33%) failed before 45°C compared with all 12:12 locusts (6/33=18%). A significantly lower percentage of 12:12 control females (0%) failed before 45°C than 16:8 control females (42%) (z-test, z=2.109, P=0.035) and 16:8 HS males (50%) (z-test, z=2.309, P=0.021) (Table 1). There were no other significant differences among groups (z-tests, P>0.05).
The ventilatory motor pattern failed before 30 min at 45°C in a significantly greater percentage of all 16:8 control locusts (19/19=100%) than all 12:12 control locusts (12/16=75%) (z-test, z=2.316, P=0.021). The ventilatory motor pattern failed before 30 min at 45°C in a significantly greater percentage of all 16:8 HS locusts (17/17=100%) than all 12:12 HS locusts (13/17=76%) (z-test, z=2.128, P=0.033). All 12:12 females, both control and HS, were significantly more likely to tolerate increasing and constant high temperature stress at 45°C compared to the other three control and HS groups, respectively (z-tests: 12:12 control females vs 12:12 control males, z=2.309, P=0.021; 12:12 control females vs 16:8 control females, z=2.739, P=0.006; 12:12 HS females vs 12:12 HS males, z=2.426, P=0.015; 12:12 HS females vs 16:8 HS females, z=2.426, P=0.015) (Table 1). Thus, 12:12 animals were significantly more likely to withstand increasing and constant high temperature stress than 16:8 animals, further evidence of improved thermotolerance.
Another important measure of thermotolerance is the percentage of locusts whose ventilatory rhythm recovered before 30 min at room temperature following hyperthermic failure (Table 1). There were some instances in which recovery of motor patterning following hyperthermic failure did not occur before 30 min at room temperature (RT) (Table 1). Of the locusts whose ventilatory rhythm failed before 30 min at 45°C, there was 100% recovery before 30 min at RT in all groups except for 12:12 control males (63%) and 16:8 control females (83%) (Table 1). There were no significant differences in percentage of recovery among groups (z-tests, P>0.05).
Effect of photoperiod, pre-treatment, and the sex of the animal on ventilatory rate at room temperature and during high temperature stress
We compared mean ventilatory frequencies at 20°C
(Fig. 4A), 40°C
(Fig. 4B), and the average
change in ventilatory frequency from 20 to 40°C
(Fig. 4C) in each group to
determine if there were effects of photoperiod, pre-treatment, or sex on these
parameters.
There were significant differences in ventilatory rate at 20°C among groups (three-way ANOVA, P=0.0006, F(7,56)=4.3823) (Fig. 4A). Statistical analysis revealed a main effect of pre-treatment on ventilatory rate at 20°C (three-way ANOVA, P=0.0425, F(1,56)=4.3108), which is difficult to interpret because there was a significant interaction between pre-treatment and sex (three-way ANOVA, P=0.0002, F(1,56)=15.5389). The main effect of pre-treatment was driven by a significant effect of pre-treatment on ventilatory rate at 20°C in females (control females vs HS females: post hoc Tukey test, P<0.05). 16:8 control females had a significantly lower ventilatory rate at 20°C than 16:8 control males, 16:8 HS males, 16:8 HS females, and 12:12 HS females (post hoc Tukey tests, P<0.05).
There were significant differences in ventilatory rate at 40°C among groups (three-way ANOVA, P<0.0001, F(7,50)=6.6099) (Fig. 4B). Statistical analysis revealed a main effect of photoperiod (three-way ANOVA, P=0.0001, F(1,50)=17.0251) and a main effect of sex (three-way ANOVA, P=0.0013, F(1,50)=11.6802) on ventilatory rate at 40°C. There were no statistically significant interactions between photoperiod, pre-treatment, and sex. 16:8 control females had a significantly lower ventilatory rate at 40°C than 16:8 HS males, 12:12 control males, 12:12 HS males, and 12:12 control females (post hoc Tukey tests, P<0.05). 16:8 HS females had a significantly lower ventilatory rate at 40°C than 12:12 HS males (post hoc Tukey test, P<0.05).
There were significant differences in the change in ventilatory rate from 20 to 40°C among groups (three-way ANOVA, P<0.0001, F(7,49)=8.6146) (Fig. 4C). Statistical analysis revealed a main effect of photoperiod (three-way ANOVA, P<0.0001, F(1,49)=26.9272) and a main effect of sex (three-way ANOVA, P=0.0028, F(1,49)=9.9118) on the change in ventilatory rate from 20-40°C, which is difficult to interpret because there was a significant interaction between sex and pre-treatment (three-way ANOVA, P=0.0018, F(1,49)=10.9587). The main effect of sex was driven by a significant effect of sex on the change in ventilatory rate from 20 to 40°C in HS locusts (HS females vs HS males: post hoc Tukey test, P<0.05). 12:12 HS males had a significantly higher change in ventilatory rate from 20 to 40°C than 16:8 control males, 16:8 control females, 16:8 HS females, and 12:12 HS females (post hoc Tukey tests, P<0.05). 12:12 control males and 12:12 control females had a significantly higher change in ventilatory rate from 20 to 40°C than 16:8 control females and 16:8 HS females (post hoc Tukey tests, P<0.05).
Effect of photoperiod, pre-treatment, and the sex of the animal on time-to-failure and time-to-recovery of the ventilatory motor pattern
We compared time-to-failure of ventilatory motor pattern generation in
response to increasing and constant high temperature stress
(Fig. 5A) and time-to-recovery
of motor patterning (Fig. 5B)
in each group to determine if there were effects of photoperiod,
pre-treatment, or sex on these parameters.
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There were significant differences in time-to-recovery among groups (three-way ANOVA, P<0.0001, F(7,48)=10.2533) (Fig. 5B). Statistical analysis revealed a main effect of photoperiod (three-way ANOVA, P<0.0001, F(1,48)=28.1415) and a main effect of pre-treatment (three-way ANOVA, P=0.0053, F(1,48)=8.5216) on time-to-recovery. These main effects are difficult to interpret because there is a statistically significant interaction between photoperiod, pre-treatment and sex (three-way ANOVA, P=0.0095, F(1,48)=7.2972). 16:8 control females had a significantly longer time-to-recovery than all other groups (post hoc Tukey tests, P<0.05) and 16:8 HS males had a significantly longer time-to-recovery than 12:12 HS males (post hoc Tukey test, P<0.05).
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There was a significant negative correlation between ventilatory frequency at 40°C and time-to-recovery of the motor pattern following failure (Pearson Product Moment Correlation, r=-0.6, P<0.0001) (Fig. 6B).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Long-term effects of photoperiod
12:12 females weighed significantly more than 16:8 females, and 12:12 males
had a significantly greater F/C ratio than 16:8 males. The
F/C ratio is a reliable indicator of phase change in locusts
(Dirsh, 1951;
Dirsh, 1953), i.e. the ability
of locusts to exhibit density dependent polymorphism in characters such as
development, morphometry, reproduction, behaviour, colour, etc. Thus,
differences in this morphometric index suggest fundamental physiological
differences in males from each photoperiod. 12:12 locusts had a higher
survival probability than 16:8 locusts, and this difference increased with
time. There were no significant differences in survival probability between
males and females within each photoperiod, indicating that the differences in
longevity were driven primarily by photoperiod. Photoperiod is a reliable
seasonal cue for insects that has been shown to modify a variety of life
history traits such as development time to adulthood
(Nylin and Gotthard, 1998),
body size (Uvarov, 1966;
Uvarov, 1977;
Tanaka and Okuda, 1996;
Nylin and Gotthard, 1998) and
sexual maturation (Uvarov,
1966; Uvarov,
1977; Tanaka et al.,
1993; Tanaka and Okuda,
1996). Trade-offs between the above life history traits and
longevity have also been examined
(Parsons, 2004;
Sorensen and Loeschcke, 2004).
African migratory locusts are naturally adapted to a 12:12 photoperiod, so a
longer daylength photoperiod of 16:8 could have inflicted a number of fitness
consequences. The 16:8 colony could be considered a stressful habitat for a
number of reasons. 16:8 animals were awake, active and exposed to light and
heat (emitted from light bulbs inside cages) for longer periods than 12:12
animals. Increased daily activity increases metabolism, and increased exposure
to light and heat results in more frequent and longer duration elevations in
core temperature, potentially inducing stress and further increasing
metabolism. Since 16:8 animals were exposed to a life-long cycle of extended
light hours, longer bouts of activity and longer periods of high temperature,
we conclude that the metabolic costs incurred resulted in accumulation of
deleterious changes in cells and tissues, manifested as aging, and a more
pronounced decrease in survival probability with time compared to 12:12
animals.
Effect of photoperiod on thermosensitivity of the ventilatory CPG
16:8 control locusts had a lower ventilatory frequency than 12:12 control
locusts at each temperature during a temperature ramp. L. migratoria
reared in long-day photoperiods (L:D 16 h:8 h) had lower oxygen consumption
rates than those reared in short-day photoperiods (L:D 12 h:12 h)
(Tanaka and Okuda, 1996). The
ventilatory response to increasing thermal stress differed as a function of
photoperiod in both control groups of locusts and HS groups of locusts. The
differences in thermosensitivity of 16:8 and 12:12 locusts could be attributed
to an effect of photoperiod on circadian rhythms, resulting in different
coordination of metabolic processes.
Circadian clocks are adaptive for insects by generating rhythms that can be
entrained to environmental cycles and/or by coordinating various internal
processes. There is evidence that ventilation and metabolism have oscillations
dependent on the time of day, suggesting that these processes are endogenously
controlled (Saiki and Mortola,
1995; Peever and Stephenson,
1997). Peaks of oxygen consumption that occurred at certain times
in a 24 h cycle and persisted during extended darkness were found in two
species of cockroaches, B. giganteus and B. craniifer
(Banks et al., 1975). A
parallel morning-night difference in ventilation and metabolism was found in
6-day-old rats and hypoxia interferes with this coupling
(Saiki and Mortola, 1995). If
similar mechanisms operate in the locust, cyclic oscillations of metabolism
and ventilation might explain differences in ventilatory rate of locusts
reared under different daylength cycles.
It is unclear how circadian systems are organized and how clocks
controlling different rhythms are related, but studies on Drosophila
have provided evidence for expression of clock genes in both the brain and
peripheral tissues (Giebultowicz,
1999). Many peripheral oscillators in insects are light-sensitive
and capable of being synchronized by the sun, resulting in any number of
independently entrained clocks that determine the physiological state of an
animal. There are two light-entrainable circadian clocks in saturnid moths,
one in the forebrain and another in the prothoracic gland, each independently
coupled to the L:D cycle (Pittendrigh,
1993). Every known oscillating tissue in Drosophila can
be reset by light (Plautz et al.,
1997). There are numerous circadian oscillators in
Drosophila, suggesting the presence of independent photoreceptive
clocks throughout the fly with light as the master coordination signal
(Plautz et al., 1997).
We presume that locusts have circadian organization and components
analogous to that of Drosophila; however, this has not been
thoroughly investigated. The rhythm of cuticle growth in locusts is light
sensitive (Neville, 1967),
suggesting an independent circadian mechanism located in epidermal cells that
is entrained to L:D hours (Giebultowicz,
1999). If circadian clocks controlling ventilatory rhythms in
locusts are synchronized by the sun, exposure to different L:D hours could
result in different metabolic and ventilatory oscillations and thus a
different resting ventilatory rate and response to increasing temperature, as
seen in our 16:8 and 12:12 locusts.
Relationship between ventilatory frequency and tolerance to high temperature stress
12:12 locusts had a higher ventilatory frequency at 40°C and change in
ventilatory frequency from 20°C to 40°C than 16:8 locusts. 12:12
locusts were also able to cope with high temperature stress and continue to
ventilate for a longer period than 16:8 locusts. We found a strong positive
correlation between time-to-failure and ventilatory frequency at 40°C such
that these two variables almost always increased together. Increased
ventilatory rate is an adaptive response to facilitate water loss and increase
evaporative cooling, and to effectively dissipate body heat in insects
(Prange, 1990;
Prange, 1996). A smaller
percentage of 12:12 animals failed during increasing and constant high
temperature stress than 16:8 animals, evidence that animals from the 12:12
colony are better able to tolerate heat and maintain neural functions
necessary for survival. We suggest that 16:8 animals were more vulnerable to
high temperature stress due to the lack of thermoregulation afforded by fast
abdominal pumping.
16:8 animals had a longer time-to-recovery than 12:12 animals, and we found that time-to-recovery almost always decreased as ventilatory rate at 40°C increases. The correlation between ventilatory rate at 40°C and time-to-recovery was not as tight as the correlation between ventilatory rate at 40°C and time-to-failure. We presume that increased ventilatory rate at high temperatures directly affected time-to-failure, whereas there does not appear to be any cooling mechanisms that could directly affect time-to-recovery, which would explain a less tight correlation. Time-to-failure and time-to-recovery were both improved in locusts that had a high ventilatory rate at 40°C, which suggests a linkage between time-to-failure and time-to-recovery, i.e. pleiotropic protective mechanisms.
Photoperiod-induced variation in the heat shock response of the ventilatory motor pattern
Although we found that 12:12 animals were better able to cope with heat
stress in general than 16:8 animals, an interesting question is why we did not
observe an overall increase in thermoprotection of the ventilatory motor
pattern following HS as seen in previous studies
(Newman et al., 2003).
Acquired thermotolerance was observed most clearly in 16:8 females, the group
that seemed to perform most poorly in the control condition compared to all
other groups. 16:8 control females had the lowest ventilatory frequency at
20°C and 40°C, and ventilatory frequency increased at these
temperatures in 16:8 HS females. 16:8 HS females were able to maintain motor
pattern generation for a longer period during high temperature stress and were
able to recover more quickly following failure compared to 16:8 control
females. HS did not improve the response of 12:12 females to subsequent heat
stress. Thus, our study demonstrates photoperiod-induced plasticity of the HS
response.
It is not well understood how a prior HS induces long-term changes in
cells, or how an organism's interaction with its environment modulates the HS
response. One aspect believed to be involved in the stress response is the
induction of Hsps. Although Hsps are molecular chaperones that alleviate
stress and play a key role in inducible thermotolerance, they are
energetically costly and can be harmful if overproduced
(Krebs and Feder, 1997;
Krebs and Feder, 1998). The
functional consequences of Hsps are concentration dependent and the
concentration depends on the level of stress in a habitat
(Feder and Hofmann, 1999),
which could very well have varied in the 16:8 and 12:12 colonies.
Neuromodulators such as serotonin are also believed to be involved in the HS
response (Hirashima and Eto,
1993; Newman et al.,
2003). Serotonin plays a role in the modulation of many
physiological processes (Osborne,
1996), and has been shown to mimic the effects of a HS on
ventilation during subsequent temperature stress
(Newman et al., 2003). Large
fluctuations in serotonin levels were shown in the locust MTG following 4 h of
crowding of solitarious animals (Rogers et
al., 2004), indicating that these types of rapid changes in
serotonin levels are possible in a short time-frame in response to important
environmental stimuli, and, moreover, in the same ganglion that houses some of
the neurons that are critical for generating the ventilatory rhythm. Serotonin
levels in the optic lobes of crickets (Gryllus bimaculatus), which
house bilaterally paired circadian pacemakers, fluctuate depending on the time
of day, regulating sensitivity of neurons
(Saifullah and Tomioka, 2002).
The involvement of serotonin in the HS response and the evidence for daily
rhythms of serotonin levels in insects could explain differences in the HS
response of our 16:8 and 12:12 locusts. Thus, it is likely that conditions
associated with daylength impose constraints on resources devoted to
thermoprotection, such as Hsps and neuromodulators, and the way in which these
are regulated.
This study provides further insight into phenotypic plasticity of neural function and the mechanisms underlying adaptation to environmental stress. We provide evidence that photoperiod can have profound effects on locusts in a number of ways. Animals reared under a photoperiod more closely resembling that of their natural habitat (L:D 12 h:12 h) are more thermosensitive and are able to maintain neural function during high temperature stress for a longer period than animals reared under a long-day photoperiod (L:D 16 h:8 h). This is likely due to improved heat loss mechanisms associated with fast abdominal pumping. 12:12 locusts live longer than 16:8 locusts, further evidence that light:dark hours can have a marked influence on insects. The effects of stress pre-treatment varied in animals reared under different photoperiods, indicating that the mechanisms underlying acquired thermoprotection are plastic. Our main conclusion is that neural circuit operation is plastic and environmental variables such as photoperiod modulate properties of neural function such that the ability to cope with stress is affected.
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