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First published online February 1, 2008
Journal of Experimental Biology 211, 531-538 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013136
Competition between immune function and lipid transport for the protein apolipophorin III leads to stress-induced immunosuppression in crickets
1 Department of Psychology, Dalhousie University, Halifax, NS B3H 4J1,
Canada
2 Institute for Marine Biosciences, National Research Council of Canada, 1411
Oxford Street, Halifax, NS, Canada
* Author for correspondence (e-mail: sadamo{at}dal.ca)
Accepted 6 November 2007
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: Orthoptera, lipophorin, flight, Gryllus texensis, trade offs, disease resistance, Serratia marcescens
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), other
vertebrates (Ewenson et al.,
2003; Thomas et al.,
2005) and insects (Adamo and
Parsons, 2006). This window of vulnerability appears maladaptive,
suggesting that it is caused by a physiological constraint. For example,
crickets (Gryllus texensis) are more likely to die from an infected
wound after flight-or-fight behaviors
(Adamo and Parsons, 2006).
During an intense fight, crickets become less able to defend themselves
against bacteria, even though intense fighting increases their chance of being
exposed to bacteria through a wound. We hypothesize that this transient
decline in disease resistance in crickets is caused, at least in part, by
physiological interactions between the immune system and lipid transport.
These two systems are intertwined in both vertebrates
(Berbée et al., 2005;
Wendel et al., 2007) and
insects (Weers and Ryan,
2006).
In insects, two proteins, apolipophorin I and apolipophorin II combine to
form high-density lipophorin (HDLp). HDLp ferries a variety of lipophilic
compounds through the blood (hemolymph) (for a review, see
Weers and Ryan, 2006). A third
protein, apolipophorin III (apoLpIII), exists as a monomer at rest, but during
energy-demanding behaviors, undergoes a conformational change and combines
with HDLp to form low density lipophorin (LDLp)
(Weers and Ryan, 2006). LDLp
can carry the large amount of lipid (diacylglycerol)
(Weers and Ryan, 2006),
liberated from the fat body, needed to fuel flight in long-winged gryllid
crickets (Zera et al.,
1999).
However, lipid-free or monomeric apoLpIII also has immunological functions.
It is thought to act as a pattern recognition molecule
(Weers and Ryan, 2006).
Apolipophorin III, like lipophorins in mammals
(Wendel et al., 2007), can
bind and detoxify lipopolysaccharides (LPS)
(Dunphy and Halwani, 1997). It
also binds to lipoteichoic acid and bacterial surfaces
(Halwani et al., 2000), as
well as to β-1,3-glucans and fungal conidia
(Whitten et al., 2004). Once
bound to pathogens or their components, apoLpIII is thought to undergo a
conformational change that activates an immune response against the pathogen
(Leon et al., 2006;
Weers and Ryan, 2006). It then
promotes cellular immune reactions such as phagocytosis
(Wiesner et al., 1997) and an
increase in antibacterial activity in the hemolymph
(Wiesner et al., 1997;
Dettloff et al., 2001a). Thus,
apoLpIII appears to act as a circulating detector for bacteria
(Kim et al., 2004).
ApoLpIII is required only intermittently for lipid transport and pathogen
defense. Therefore, most of the time, these functions are not in conflict.
However, because both lipid and LPS bind to the same position on apoLpIII
(Leon et al., 2006), we
hypothesize that apoLpIII cannot carry out both of its functions
simultaneously. Once co-opted into lipid transport, it may no longer be
available for immune surveillance. Reduced immune surveillance could explain
the appearance of the transient period of immunosuppression that occurs
immediately after flight-or-fight behavior
(Adamo and Parsons, 2006).
To test this hypothesis, we first determined whether apoLpIII is depleted
by both flying and an immune challenge in the cricket G. texensis,
demonstrating the potential for conflict between lipid transport and immune
function. We then tested whether disease resistance is related to the level of
free apoLpIII in the hemolymph by reducing apoLpIII concentration using
adipokinetic hormone (AKH). AKH mobilizes lipid in the cricket Acheta
domesticus (Woodring et al.,
2002) and induces the formation of LDLp
(Strobel et al., 1990).
Finally we injected apoLpIII and tested whether we could prevent
flight-induced immunosuppression by increasing apoLpIII levels.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)] were
originally collected near Austin, Texas and have been maintained as a
laboratory colony for many generations. Crickets were reared at
28±2°C on a 12 h:12 h L:D cycle. Experiments were run at
approximately the same time each day to avoid any circadian rhythm effects in
hemolymph lipid levels (Das et al.,
1993). Pellets of dry cat food and water were provided ad
libitum. No cricket was used in more than one experiment. Tests were
performed approx. 2 weeks (±3 days) after the molt to adulthood. At
this age crickets are sexually mature and within their lifespan in the field
(Murray and Cade, 1995). It is
also well before the immune system begins to decline due to senescence
(Adamo et al., 2001). Hemolymph was removed by puncturing the pronotal membrane with a 10 µl Hamilton syringe needle and collecting 2 µl of hemolymph. Injections were also given through the pronotal membrane unless hemolymph was to be subsequently withdrawn. In that case, injections were given into the abdomen between the third and fourth caudal tergite.
Crickets used in experiments were isolated into individual opaque containers (10 cm in diameter) 1 day prior to use with food and water provided ad libitum. Unless otherwise indicated, groups were matched for sex.
All studies were approved by the Animal Care Committee of Dalhousie University and are in accordance with Canadian Council on Animal Care. All chemicals were from Sigma-Aldrich (St Louis, MO, USA) unless otherwise noted.
Flying crickets
To test the effect of flight on immune function, crickets were tethered to
a wooden applicator stick using low temperature wax and placed in a gentle air
stream. Crickets were allowed to fly for 5 min. Control crickets were
unhandled. Therefore, flying crickets experienced both a handling stress and a
flight stress. Earlier work (Adamo and
Parsons, 2006) demonstrated that physical activity, as opposed to
handling stress, is required to produce immunosuppression in G.
texensis. Crickets that did not fly during the trial were excluded from
the study. In locusts, 5 min of flight activates the mobilization of energy
reserves (Auerswald and Gäde,
2006), including the release of lipid 1 h after flight. Auerswald
and Gäde (Auerswald and Gäde,
2006) hypothesize that after 5 min of flight, animals are
hormonally committed to release lipid.
Measurement of total lipid content of the hemolymph
The effect of flight on total hemolymph lipid concentration was measured
using the sulfuric-vanillin method (Barnes
and Blackstock, 1973). 2 µl of hemolymph was added to 500 µl
of 95% sulfuric acid heated to 95°C. Samples were heated for 10 min and
cooled to room temperature on ice. 200 µl was then added to 1 ml vanillin
agent (1.98 g vanillin, 668 ml orthophosphoric acid, 332 ml water). After 15
min, spectrophotometric readings were taken at 540 nm. Samples were run as
duplicates. Diacylglycerol was used as the lipid standard. Standards were run
with each trial.
Identification of free ApoLpIII using native PAGE and SDS-PAGE gels
ApoLpIII was identified using the methods of Smith et al.
(Smith et al., 1994). ApoLpIII
has little sequence homology among different orthopteran species
(Strobel et al., 1990), and
therefore sequence homology cannot be used to positively identify the band.
Identification of apoLpIII was based on molecular mass and by the dramatic
decline in the density of the band in response to adipokinetic hormone
(Smith et al., 1994).
Hemolymph from 2 crickets (4 µl total) was pooled in 20 µl of loading
buffer with cricket anti-coagulant (loading buffer: 0.5 mol
l–1 Tris–HCl pH 6.8, 10% SDS (w/v), 25% glycerol, 0.2%
Bromophenol Blue; cricket anti-coagulant: phenothiocarbamide and protease
inhibitor cocktail (a few crystals enough to form a supersaturated solution),
10 mmol l–1 EDTA, 0.15 mol l–1 NaCl, 10 mmol
l–1 glutathione) kept on ice. The hemolymph–loading
buffer mixture was spun at 2500 g for 10 min at 4°C. The
supernatant was added to a 7% native-PAGE gel with 1 mmol l–1
EDTA. After running the sample on the native-PAGE gel, columns were cut and
placed horizontally on top of a 12% SDS-PAGE gel. Gels were stained with
silver stain (Swain and Ross,
1995). Molecular mass markers (Bio-Rad, Hercules, CA, USA) were
run with the hemolymph samples.
To determine the effects of different treatments on the relative amount of
free apoLpIII in the hemolymph, the darkness and size of the apoLpIII band was
calculated using NIH Image software (ImageJ 1.38x). The darkness of this band
was compared to the average darkness of two prominent unidentified bands (see
Fig. 1, bands 1 and 2; 
65
kDa and 75 kDa) that did not differ in intensity depending on treatment (i.e.
flying, immune challenge, AKH injection or pre-loading with trehalose;
Kruskal–Wallis=2.6, P=0.45). The ratio of the darkness of the
apoLpIII band was divided by the average darkness of bands 1 and 2 (see
Fig. 1) in each gel and this
ratio (band darkness ratio) was used to compare the relative amount of
apoLpIII across samples. The amount of free apoLpIII in the hemolymph was
quantified by comparing the darkness of bands of dilutions of known amounts of
purified apoLpIII (see below) with the apoLpIII bands in the hemolymph.
ApoLpIII standards were run on the same gel as the hemolymph samples.
|
). Although
individual immune assays can provide complimentary information, such assays
are difficult to interpret without first establishing whether there is a
biologically significant change in immune function (i.e. disease resistance).
S. marcescens is a Gram-negative bacterium found world-wide in both
soil and water and has been recovered from the bodies of crickets in the field
(Steinhaus, 1959). The
bacterium is not lethal unless it enters the hemocoel
(Steinhaus, 1959). We gave an
LD50 dose of S. marcescens by injecting crickets with
approx. 1x104 cells per 2 µl in culture medium. Initially
we determined bacterial cell number using a Petroff-Hauser cytometer and
phase-contrast microscopy. Later bacterial cell number was estimated
spectrophotometrically. We obtained bacteria from Carolina Biological Supply
Co. (Burlington, North Carolina, USA). Injected crickets were housed
individually in opaque containers (diameter 10 cm) with food and water ad
libitum. We recorded mortality daily for the next 4 days after the
bacterial injection, because mortality due to S. marcescens occurs
within 4 days of injection (Adamo et al.,
2001).
Effect of flight and immune challenge on total lipid content and free ApoLpIII concentration of the hemolymph
To assess the effect of flight on total lipid concentration in the
hemolymph, we tethered crickets and allowed them to fly for 5 min. Control
crickets remained unhandled during this period. 2 µl of hemolymph was taken
from control and flying crickets 15, 60, 90 or 120 min after the 5 min flight.
Times were based on those of Woodring et al.
(Woodring et al., 2002). Blood
was taken only once from each cricket.
To determine the effect of immune challenge on hemolymph lipid
concentrations, 2 µl of hemolymph was removed from crickets 90 min after
injection of either heat-killed S. marcescens (1x105
cells) or sterile nutrient broth. Heat killed S. marcescens showed no
growth when placed on agar plates. Injecting heat-killed S.
marcescens induces an immune response in G. texensis
(Adamo, 2004b). The time point
of 90 min was chosen based on preliminary results and values in the literature
(Mullen et al., 2004).
To determine the effects of flight on free apoLpIII levels, crickets were tethered and flown for 5 min. Crickets were then returned to their individual containers and 2 µl of hemolymph was collected from flown and control crickets 1 h later. The timing was based on the results of the effect of flight on lipid hemolymph levels (Fig. 2). The hemolymph was then run on a modified two-dimensional gel (native and SDS-PAGE) as described above, to determine the relative amount of free apoLpIII compared to controls.
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Effect of adipokinetic hormone on total lipid, free ApoLpIII concentration and on resistance to S. marcescens
To test the effect of adipokinetic hormone (AKH; from Gryllus
bimaculatus; Bachem, Bubendorf, Switzerland) on the lipid concentration
in the hemolymph of G. texensis, we injected 20 pmol into crickets
and measured the lipid content in 2 µl of hemolymph using the method
described above. Control crickets were injected with 2 µl of the vehicle
(80% methanol). Hemolymph was collected 90 min after injection. Using the
procedures described above, hemolymph was tested for total lipid content and
free apoLpIII levels.
To test the effect of AKH on disease resistance, crickets were given an injection of 20 pmol AKH followed by an LD50 dose of S. marcescens. The AKH solution was passed through a sterile 0.2 µm filter prior to injection. Hamilton syringes were cleaned with disinfectant prior to use.
Effect of pre-loading with trehalose on total lipid, free ApoLpIII concentration and on resistance to S. marcescens
Trehalose inhibits the release of lipid from the fat body in locusts
(Thompson, 2003). Therefore,
we preloaded crickets with trehalose prior to flight to reduce the release of
lipid. To test whether trehalose can reduce the increase in lipid and the
decrease in apoLpIIII that occurs after flight, crickets were randomly
assigned to one of three groups. The first group was injected with 5 µl of
trehalose (0.5 g ml–1 insect Ringer) just prior to being
tethered and flown for 5 min. Insect Ringer was composed of 121 mmol
l–1 sodium chloride, 4.1 mmol l–1 CaCl, 1.37
mmol l–1 dibasic potassium phosphate, 198 µmol
l–1 monobasic potassium phosphate, and 38.6 mmol
l–1 Tris–HCl, adjusted to pH 7.4. The second group were
injected with 5 µl insect Ringer prior to flight. The third group,
unhandled controls, were neither injected nor flown. Crickets had 2 µl of
hemolymph removed 60 min (for total lipid measurement) or 90 min (for apoLpIII
measurement) after they stopped flying. Total lipid and free apoLpIII
concentration were assessed as described above. Prior to injection, the
solution to be injected was passed through a sterile 0.2 µm filter.
Hamilton syringes were cleaned with disinfectant prior to use.
To determine whether trehalose could prevent the decline in disease resistance in flying crickets, crickets were randomly assigned to four groups. The first two groups were injected with either trehalose or Ringer prior to flight, as described above. The second two groups were also injected with either trehalose or Ringer, but these two groups of crickets were not flown. After the 5 min flight, crickets were injected with an LD50 dose of S. marcescens. Crickets that were not flown were also injected with bacteria.
The effect of pre-loading with ApoLpIII on flight-induced immunosuppression
To determine whether injecting apoLpIII could reverse flight-induced
immunosuppression, apoLpIII was isolated from cricket hemolymph using a
procedure modified from published methods
(Mullen and Goldsworthy, 2003;
Halwani and Dunphy, 1999).
Hemolymph was collected by making a shallow incision in the pronotal membrane
and removing the hemolymph that welled up from the wound (approx. 10 µl).
Hemolymph was immediately placed in an ice-cold microcentrifuge tube
containing cricket anti-coagulant (0.15 mol l–1 NaCl, 10 mmol
l–1 EDTA, 10 mmol l–1 glutathione, and a few
crystals of phenylthiocarbamide and protease inhibitor cocktail). The
anti-coagulant:hemolymph ratio was approx. 5:1). The hemolymph mixture was
heated at 96°C for 5 min and then spun at 10 000 g for 5
min at 4°C. The supernatant was removed and stored at –20°C. The
pellet was washed once and the wash was also collected. Pellets, wash and
supernatant were shipped to Guild Biosciences (Charleston, South Carolina,
USA) for apoLpIII purification. Briefly, heat treated supernatant was buffer
exchanged by gel filtration using a Zeba 10 ml column equilibrated with 160
mmol l–1 ammonium acetate pH 6.5. The material was then
partially purified by passing it through an Econo-Pac Q ion exchange column
equilibrated with 160 mmol l–1 ammonium acetate (pH 6.5)
using a Waters HPLC system with the separation monitored by absorbance at 280
nm. The flow through was collected and its purity was assessed by SDS-PAGE
with silver staining. The flow-through material was concentrated using an
Ultra 4 centrifugal concentrator then diluted with 0.4% w/v CHAPS containing
50 mmol l–1 Hepes pH 6.5. The material was then fractionated
by size exclusion chromatography using a Zorbax GF-250 (4.6x250 mm, 4
µm) column equilibrated with 0.4% w/v CHAPS with 50 mmol
l–1 Hepes pH 6.5 on a Waters HPLC system. Fractions were
assessed by SDS-PAGE with silver staining
(Swain and Ross, 1995).
Protein concentration was determined using the Bradford assay with bovine
albumin as the standard (Bradford,
1976).
Crickets were randomly assigned into four groups. The first group was
injected with 15 µl apoLpIII (5 µg apoLpIII µl–1
insect Ringer). We used this concentration because it was above the amount
required by Dettloff et al. (Dettloff et
al., 2001a) to increase immune function. Control injected crickets
(group 2) were injected with 5 µl insect Ringer. Group 3 was injected with
2 µl of heat-killed S. marcescens prior to flight. The
concentration injected is sufficient to induce an immune reaction in G.
texensis (Adamo, 2004b).
Crickets were then flown for 5 min. Unhandled controls (group 4) were neither
injected nor flown. After the 5 min flight, crickets in all groups were
injected with an LD50 dose of S. marcescens. Control crickets were
injected at the same time, even though they were not flown.
Measurement of hemolymph volume
Hemolymph volume was measured using a modified version of the procedure of
Ehler et al. (Ehler et al.,
1986). Radioactive [14C]inulin (Perkin Elmer, Waltham,
MA, USA) was diluted in insect Ringer resulting in 56 000 d.p.m. per 5 µl.
Crickets were given a 5 µl injection into the abdomen using a 10 µl
Hamilton syringe. Injections were given between the third and fourth last
abdominal tergites, 2 mm above the abdominal spiracles. 1 h after the
injection (Ehler et al.,
1986), 2 µl of hemolymph was collected through the pronotal
membrane using a 10 µl Hamilton syringe. The hemolymph was added directly
to 5 ml of scintillation fluid and gently vortexed for 5 s. Radioactivity was
measured using a Winspectral 1414 scintillation counter (Perkin Elmer). Values
were compared to both internal and external calibration curves. To measure the
effect of 5 min of flight on hemolymph volume, crickets were injected with
inulin, and after a 5 min rest period were flown for 5 min. Hemolymph was
collected 50 min after the end of the flight. Control crickets remained in
their containers for the entire 60 min period after their inulin injection.
All crickets were weighed 2 h prior to the inulin injection. Crickets were
matched by weight and assigned to either the flying or control group.
Statistics
Data were analyzed using Prism4 (Graphpad Software Inc.) software. When
multiple comparisons were performed on the same data set, the alpha criterion
was adjusted accordingly (Sokal and Rohlf,
1981). Non-parametric analyses were carried out according to
Meddis (Meddis, 1984). Unless
noted otherwise, values in the text are means ± standard deviation
(s.d.).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) and
Gryllus bimaculatus (Tanaka et
al., 1999). The apoLpIII concentration in the hemolymph of
Gryllus texensis was 1.8±0.6 mg ml–1
(N=8). Flight increased the amount of total lipid in cricket hemolymph 60 min later (ANOVA, F(4,54)=4.0, P=0.007, Bonferroni post-hoc test, P<0.05; Fig. 2). Injection of heat-killed S. marcescens also resulted in an increase in hemolymph lipid concentration (7.8±2.4 mg ml–1 hemolymph, N=13) relative to that in nutrient broth-injected animals (6.7±2.7 mg ml–1, N=14), 90 min after injection (t(25)=2.2, P=0.03). Both flight (Mann–Whitney, U=16.0, P=0.001, N=12/group, 46% decrease from controls) and an immune challenge (Mann–Whitney, U=21.5, P=0.03, N=10/group, 63% decrease from controls) resulted in a decline in the amount of free apoLpIII in the hemolymph compared to controls. The increase in lipid during an immune challenge is less than that observed during flight (t(22)=2.6, P=0.02). However, the reduction in free apoLpIII levels is greater during an immune challenge than it is during flight (Mann–Whitney, U=32.5, P=0.075).
Injecting AKH into G. texensis induced an increase in hemolymph lipid concentration (Fig. 3A, t(7)=2.8, P=0.03) and a decrease in apoLpIII concentration relative to controls (Fig. 3B; Mann–Whitney, U<0.0001, P=0.03, N=4). AKH also produced a decline in resistance to the bacterium Serratia marcescens relative to vehicle-injected controls (Fig. 3C; Fisher's exact test, P=0.04).
|
),
L=108, P=0.001]. Crickets preloaded with trehalose survived
a post-flight challenge with S. marcescens better than those
preloaded with the vehicle [i.e. insect Ringer;
Fig. 4C; test for trends
(Meddis, 1984)
Z=2.53, P<0.01]. Trehalose injections alone did not
increase survival of a bacterial challenge in crickets that were not flown
(Fig. 4C), demonstrating that
trehalose itself does not enhance resistance to S. marcescens.
|
Preloading crickets with apoLpIII prior to flight reduced immunosuppression (Fig. 5). An immune challenge did not enhance survival (Fig. 5).
|
). Therefore,
changes in lipid and free apoLpIII concentration observed after flight were
not due to changes in blood volume. |
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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After delivering lipid, LDLp is thought to release apoLpIII back into the
hemolymph (Weers and Ryan,
2006) suggesting that lipid transport need not have a large impact
on apoLpIII concentrations. Nevertheless, others have also found that AKH
and/or an immune challenge result in a measurable decline in free apoLpIII
concentration (Mullen and Goldsworthy,
2003; Mullen et al.,
2004). Up to 16 molecules of apoLpIII can associate with one
particle of HDLp to form LDLp (Weers and
Ryan, 2006). Therefore, even a small increase in LDLp could reduce
apoLpIII concentrations.
The increase in disease resistance that we observed after an injection of
apoLpIII supports earlier work that showed that injected apoLpIII increases
antibacterial activity in the hemolymph of other insects (Lepidoptera) (e.g.
Wiesner et al., 1997;
Halwani and Dunphy, 1999;
Kim et al., 2004). Wiesner et
al. (Wiesner et al., 1997)
found that the greater the amount of apoLpIII injected, the greater the
increase in antibacterial activity in the hemolymph. This result supports our
hypothesis that the amount of apoLpIII in the hemolymph determines, in part,
the ability of the immune system to respond to bacterial challenges. Our
apoLpIII results were not due to non-specific immune activation. Activating
the immune system by injecting heat-killed S. marcescens
(Adamo, 2004b) did not have a
protective effect (Fig. 5). We
do not know the fate of apoLpIII after we injected it. It is possible that it
formed LDLp, and it was the LDLp that was responsible for the immune-enhancing
effect (Dettloff et al.,
2001a; Dettloff et al.,
2001b). However, Halwani and Dunphy
(Halwani and Dunphy, 1999)
argue that apoLpIII remains in its native state when injected. Moreover,
injections of LDLp do not enhance antibacterial activity in the hemolymph
(Dettloff et al., 2001b),
suggesting that it would not compensate for the loss of bacterial resistance
induced by flight. Furthermore, flight results in the generation of LDLp
(Chapman, 1998), and,
therefore, if LDLp levels were critical for bacterial resistance, flight would
not be expected to be immunosuppressive.
Paradoxically, AKH injections result in enhanced phenoloxidase (PO)
activity in response to an immune challenge in locusts
(Goldsworthy et al., 2003),
despite the fact that it also reduces apoLpIII levels
(Mullen and Goldsworthy, 2003)
and decreases disease resistance
(Goldsworthy et al., 2005).
These results suggest that the immune-enhancing effects of AKH are smaller in
magnitude than the immune-suppressing effects of AKH-induced apoLpIII
reduction. We speculate that the function of the enhancing effects of AKH on
phenoloxidase activity may be to help maintain adequate immune function
despite the reduction in apoLpIII levels.
Trehalose not only inhibits the release of lipid, it also delays the
release of AKH (Thompson,
2003). Therefore, it is possible that the effect of preloading
flying crickets with trehalose was caused by reducing the effects of AKH on
immunity and not by suppressing the decline in free apoLpIII. For example, AKH
can reduce protein synthesis
(Kodrík and Goldsworthy,
1995) and this may have immunosuppressive effects. However, if AKH
were directly responsible for flight-induced immunosuppression, then
injections of apoLpIII should not have reversed the effect of flight.
If our hypothesis is correct, an immune challenge occurring prior to flight
should reduce an orthopteran's flying ability because of a reduction in lipid
transport capacity. This would not be an energetic constraint per se.
The animal may have substantial fat stores, but still run into an `energy'
shortage because of an inability to mobilize those stores due to a lack of
apoLpIII. As predicted, immune challenged locusts show a decrease in flight
ability (i.e. reduced flight time) that can be reversed by injecting trehalose
(Seyoum et al., 2002).
Moreover, locusts infected with fungus appear to have a decreased ability to
raise their lipid levels in response to AKH (given as an extract of the
corpora cardiaca) than do controls (Seyoum
et al., 2002). This result is consistent with the hypothesis that
as apoLpIII binds with fungal compounds, less is available to form LDLp.
Therefore, less lipid would be able to enter the hemolymph during flight or in
response to AKH. Other explanations are possible however, including a
manipulative effect of the fungus (Seyoum
et al., 2002).
ApoLpIII is only one of many pattern recognition molecules that exist in
insects (Kanost et al., 2004).
Why the decline in this particular molecule leads to such a strong deficit in
disease resistance is unclear. Part of this confusion is due to our lack of
understanding of the precise role apoLpIII plays in immune function. In fact,
the roles of apoLpIII, LDLp and HDLp in immune function are still being
assessed in insects (Zakarian et al.,
2002; Mullen and Goldsworthy,
2003; Whitten et al.,
2004; Park et al.,
2005; Leon et al.,
2006; Ma et al.,
2006; Rahman et al.,
2006). It is generally agreed that the immune system is activated
when apoLpIII changes in configuration (e.g.
Leon et al., 2006). However,
it remains unclear how the immune system differentiates between the
conformational changes that occur when apoLpIII is bound to lipid as part of
LDLp and when apoLpIII is bound to LPS
(Leon et al., 2006). It seems
unlikely that the immune system is activated every time lipid is mobilized.
Immune activation entails serious costs, including immunopathology (i.e.
self-destruction) if inappropriately deployed
(Sadd and Siva-Jothy, 2006).
Leon et al. (Leon et al.,
2006) speculate that there may be different protein conformations
depending on what the protein is bound to. This issue deserves serious
attention from insect immunologists.
Part of the difficulty in determining the immune functions of apoLpIII,
HDLp and LDLp, is that they are probably to some extent species specific
(Pratt and Weers, 2004). Their
roles may vary depending on whether an insect relies on lipid, carbohydrates
or other compounds as its major source of energy to fuel flight-or-fight
behavior. For example, whether flight-or-fight behavior is immunosuppressive
is likely to vary across, or even within, species. For example, AKH does not
induce the formation of LDLp in solitary locusts because solitary locusts
contain very low amounts of triacylglycerides in their fat body
(Chino, 1997). In this case we
would predict that injections of AKH will not be immunosuppressive in these
animals. Other insects may have different points of conflict between lipid
metabolism and immunity. For example, in some insects, lipophorin transports
carotenoids and hydrocarbons as well as lipid
(Arrese et al., 2001). Using
the same molecule for both transport and immune function is likely to produce
physiological constraints, but the exact nature of these constraints may vary
across species.
Similarly, which behaviors will produce a decline in apoLpIII and disease
resistance is likely to depend on the species. In crickets, we predict that
any behavior that results in an increase in circulating octopamine and AKH
(and hence lipid) will produce a decline in apoLpIII and disease resistance.
In crickets, fighting produces an increase in neurohormonal octopamine, but
brief escape runs do not (Adamo et al.,
1995). The fact that fighting reduces disease resistance
(Adamo and Parsons, 2006), but
brief escape runs do not (S.A.A., unpublished observations), supports our
hypothesis. Furthermore, the band darkness ratio did not differ between
unhandled controls during the trehalose trial and the vehicle-injected
controls in the AKH trial (t(5)=0.66, P=0.47).
These results suggest that injection stress does not reduce apoLpIII
concentrations. In crickets, only prolonged, physically intense behaviors are
likely to induce a reduction in apoLpIII and disease resistance.
Immune challenge has been shown to induce a lipid increase and LDLp
formation in other insects (e.g. locusts), although how the additional lipid
is released is still unknown (Mullen et
al., 2004). The increase in lipid may be needed to fuel the immune
response (Dettloff et al.,
2001b). In insects there is evidence that immune activation is
costly (Siva-Jothy et al.,
2005). However, in crickets, the increase in hemolymph lipid that
occurs during an immune challenge appears insufficient to fully explain the
decline in free apoLpIII levels. For example, the increase in hemolymph lipid
is higher during flight than during an immune challenge, but the decrease in
free apoLpIII is larger during an immune challenge. We suspect that the
pronounced decline in apoLpIII levels during an immune challenge was produced
not only by increased LDLp formation, but also by the binding of apoLpIII to
bacterial components (Pratt and Weers,
2004). This issue requires further study.
These results demonstrate how different physiological systems can `borrow'
molecules from each other to serve intermittent needs. This perspective could
help explain phenomena in a wide range of animals. For example, in
vertebrates, lipoproteins [e.g. high density lipoprotein (HDL)] are important
for both lipid (cholesterol) transport and for sequestering bacterial
lipopolysaccharides (LPS) (Berbée
et al., 2005; Wendel et al.,
2007). During a bacterial challenge, the lipoprotein composition
of HDL changes and reverse cholesterol transport declines
(Wendel et al., 2007). At
present, it is not known why the composition of HDL changes during a bacterial
challenge. If the principle in this study applies to this question, then the
changes in HDL lipoprotein composition may occur because they help shift the
function of HDL from transporting cholesterol to participating in an immune
reaction. The inability of HDL to simultaneously transport cholesterol and
bind to LPS may explain, in part, why bacterial infection can accelerate
atherosclerosis.
These results also demonstrate how multifunctional molecules can produce unsuspected trade offs. For example, the competition between lipid transport and immune surveillance for lipoproteins will result in trade offs between flight-and-fight behaviors and disease resistance in many insects. Long distance flight, male–male competition, and courtship (e.g. singing in crickets) may all exact a cost in terms of lowered disease resistance even in animals with substantial energy reserves.
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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