Substantial changes in central nervous system neurotransmitters and neuromodulators accompany phase change in the locust
1 Department of Zoology, University of Cambridge, Downing Street, Cambridge
CB2 3EJ, UK
2 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1
3PS, UK
3 Laboratory of Cognitive and Developmental Neuroscience, Babraham
Institute, Babraham, Cambridge CB2 4AT, UK
* Author for correspondence (e-mail: Smr34{at}cam.ac.uk)
Accepted 12 July 2004
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Summary |
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Key words: desert locust, Schistocerca gregaria, phase transition, HPLC, solitarious, gregarious, polymorphism
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Introduction |
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Locusts undergo an extreme form of phenotypic plasticity that is driven by
population density, which results in extensive but reversible changes in many
aspects of morphology, physiology and behaviour
(Uvarov, 1966;
Simpson et al., 1999
). Locusts
in the wild usually exist in the solitarious phase under low population
densities of less than 3 per 100 m2. Solitarious locusts are
cryptic in appearance and behaviour, fly mainly at night and actively avoid
contact with each other. If environmental conditions force locusts together
into high population densities, however, they transform to the gregarious
phase. In this phase locusts are highly active, have bright warning colours as
nymphs, are predominately day flying and, critically, are attracted towards
other locusts, thus forming cohesive groups. The rates of change of phase
characteristics vary over time scales ranging from hours to generations.
Solitarious locusts behave fully gregariously within just 4 h of crowding,
thereby increasing their propensity to move towards other locusts
(Roessingh et al., 1993
;
Roessingh and Simpson, 1994
).
By contrast, gregarious-phase locust nymphs that are isolated only partially
solitarise within 24 h, and then remain in this transitional behavioural state
for the rest of the stadium. Further behavioural solitarisation requires
isolation for several stadia (Roessingh
and Simpson, 1994
) or generations via a maternal
influence over embryonic development (Islam et al.,
1994a
,b
;
Bouaichi et al., 1995
).
The necessary sensory stimuli that trigger the initial behavioural
gregarization of solitarious locusts have been characterised
(Roessingh et al., 1998;
Hägele and Simpson, 2000
;
Simpson et al., 2001
;
Rogers et al., 2003
). There
are also clear differences in specific neuronal circuits and muscular systems
between the two extreme locust phases that can be related to these differences
in behaviour (Matheson et al.,
2003
,
2004
;
Blackburn et al., 2003
;
Fuchs et al., 2003
). The
neuro-hormonal mechanisms that drive and maintain phase change, however,
remain largely unknown (Pener,
1991
; Pener and Yerulshami,
1998
; Breuer et al.,
2003
). The peptide [His7]-corazonin promotes gregarious
colouration and morphometric changes in solitarious locusts
(Tawfik et al., 1999
;
Hoste et al., 2002
) but has no
effect on phase-related behaviour (Hoste
et al., 2002
). Previous work has analysed amounts of octopamine in
Locusta migratoria
(Fuzeau-Braesch and David,
1978
; Fuzeau-Braesch and
Nicholas, 1981
) and a partially phase-changing species,
Schistocerca americana (Morton
and Evans, 1983
), with conflicting results. No previous study has
monitored changes in neurochemicals during the phase change process from hours
to generations as we show here. As the detailed time course of behavioural
phase change is now well established, the present study analyses the
accompanying changes in putative neuromodulators and neurotransmitters within
the nervous system on a temporal scale that maximises the likelihood of
discovering coincident and hence potentially causal relationships between
changes in behaviour and neurochemistry. We used high performance liquid
chromatography (HPLC) to analyse changes in 13 different potential
neurotransmitters and neuromodulators in the central nervous system of desert
locusts at nine key stages during solitarization and gregarization. We
identify chemicals that differ quantitively between phases and track the time
course of these differences as phase-change occurs.
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Materials and methods |
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Experimental treatments
The time-course analysis of the effects of isolation and crowding was
performed by taking gregarious-phase locusts from the stock culture, isolating
different cohorts for sequentially longer periods, then taking third
generation solitarious locusts and crowding them, as shown in
Fig. 1. Nine stages of
isolation/crowding were examined in final instar nymphs. These were: (1)
long-term gregarious-phase locusts taken from the main culture, (2)
gregarious-phase locusts isolated for 24 h, (3) gregarious-phase locusts
isolated from the start of the penultimate nymphal stadium until their final
nymphal stadium, (4) first-generation, isolated-reared locusts (i.e. locusts
hatched from separated eggs and reared separately), (5) second-generation,
isolated-reared locusts (i.e. offspring of locusts reared under the previous
treatment conditions), (6) third-generation isolated-reared locusts, (7)
third-generation, isolated-reared locusts crowded together (in a group of 12)
in a standard solitarious locust-rearing cage (10 cmx10 cmx25 cm)
for 4 h, (8) third-generation isolated-reared locusts crowded together as in
(7) for 24 h and (9) third-generation isolated-reared locusts crowded together
as in (7) from the start of the penultimate larval stadium until their final
nymphal stadium.
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Each treatment group initially consisted of 12 locusts, split approximately evenly between sexes, but some losses during the experiment meant that final sample sizes ranged from 10 to 12. All locusts were analysed 25 days from their previous moult.
For comparison of neurotransmitters and neuromodulators in adult solitarious and gregarious locusts, nine gregarious locusts taken from the gregarious culture were compared with nine locusts that had been reared in isolation for two generations. All adult locusts were in the pre-reproductive stage, 510 days after their final moult.
Preparation of samples
Experimental locusts were removed from their rearing cages and placed in
7.5 cm diameter plastic plant pots, either individually if previously
isolated, or as a group if previously crowded. Some cut wheat seedlings were
added and the pots covered with pierced cling-film. The locusts were then left
undisturbed for 1 h. At the end of this resting period the pots were gently
lifted with 30 cm long forceps and plunged into liquid nitrogen; the holes in
the base of the plant pots allowed rapid access of the freezing liquid. The
frozen locusts were removed individually, decapitated, and the head and body
placed on pre-chilled dissecting blocks kept on ice. The whole brain including
the optic lobes was dissected from the head, and the complete thoracic
ganglion chain of final instar locusts, or the pro- and metathoracic ganglia
only of adult locusts, was dissected from the thorax. Ice-cold ultrapure
locust saline prepared using AnalarTM quality reagents and ultrapure
deionised water was used as necessary during the dissection. The optic lobes
were detached from the rest of the brain and the heavily pigmented retina
removed and discarded. The two optic lobes were combined to make one sample.
The central region of the brain constituted another sample and in final instar
nymphs the thoracic ganglion chain the third. In adult locusts the pro- and
metathoracic ganglia only were made into separate samples. Individual tissue
samples were placed in chilled 100 µl micro-homogenisers with 50 µl of
150 mmol l1 perchloric acid containing 100 ng
ml1 3,4-dihydroxybenzylamine hydrobromide (DHBA; Aldrich,
Poole, Dorset, UK) as an internal standard for the HPLC and homogenised for 2
min. The samples were then transferred to 1.5 ml Eppendorf tubes and
centrifuged for 30 min at 17 500 g. The supernatant was
measured using a 50 µl Hamilton syringe, transferred to another Eppendorf
tube and then stored at 80°C until the HPLC analysis (for
approximately 2 weeks).
Preparation for HPLC
Samples were analysed using three different HPLC systems designed to
measure either amino acids, monoamines or acetylcholine/choline. For the amino
acid analysis, 2 µl of the sample solution was mixed with 48 µl of
ultrapure locust saline (25x dilution); for the monoamine system, 13
µl of the sample was used undiluted, and for the acetylcholine system, 5
µl of the sample was mixed with 95 µl of ultrapure saline (20x
dilution). Standard solutions were used to calibrate each of the HPLC systems,
for both the identification and quantification of different peaks. Standard
solutions for the amino acid system contained aspartate, glutamic acid,
citrulline, glycine, arginine, taurine and -amino butyric acid (GABA),
all 250 nmol l1; for the monoamine system, octopamine (OA,
50 ng), tyramine (TA, 40 ng), DHBA (internal standard; 0.25 ng),
N-acetyldopamine (NADA, 0.2 ng), dopamine (DA, 0.5 ng) and serotonin
(5-hydroxytryptamine, 5-HT, 0.75 ng), all measured in mass per 10 µl
injected sample; and for the acetylcholine system, choline and acetylcholine
both 200 nmol l1.
Amino acid analytical system
Amino acids were analysed using an HPLC gradient system at a flow rate of
520 µl min1 (125 gradient pump; Beckman, Fullerton, CA,
USA) with a C18 reversed phase column (3 µm SphereClone column, Phenomenex,
Macclesfield, Cheshire, UK; 15 cm lengthx3.2 mm i.d., heated at
35°C) and fluorescence detection (CMA/280) as previously described
(Kendrick et al., 1996). A
Gilson (Villiers-le-Bel, France) model 231/401 auto-injector was used with
programmable precolumn derivatisation using OPA (o-pthaldialdehyde).
Injection volumes were 13 µl including both sample and OPA. Gradients and
data collection were controlled using Beckman 32 Karat HPLC software.
Detection limits were 15 nmol l1.
Monoamine analytical system
Monoamines were analysed using an isocratic HPLC system (M480 pump;
Gynkotek, Germering, Germany; flow rate 200 µl min1) with
electrochemical detection (Waters M469, Waters Milford, MA, USA, using a BAS 6
mm Unjet cell at +0.65 V) as previously described
(Kendrick et al., 1996). A
reversed phase C18 column was used (Phenomenex 3 µm SphereClone; 15 cm
lengthx2.0 mm i.d., heated at 35°C). A cooled autoinjector (CMA/200)
was used to load samples (10 µl sample volume injected). Data were
integrated using a Gynkosoft (Dionex, Sunnyvale, CA, USA) integration package.
Detection limits were 525 pg ml1.
Acetylcholine analytical system
Acetylcholine/choline were analysed using an isocratic HPLC system (CMA 250
pump, 120 µl min1) with electrochemical detection (BAS
LC4C with 6 mm Unijet cell at +0 V coated with peroxidase to produce a `wired
enzyme detector') as previously described
(Kendrick et al., 1996). A
Unijet analytical column was used (BAS, ACh/Ch column, 52 cm lengthx1 mm
i.d.). Data were integrated using a Gynkosoft (Dionex) integration package.
Detection limits were 0.5 nmol l1.
Statistical analyses of the data were made using SPSS (version 11). Outlying data points lying more than 2.5 standard deviations from the sample mean (in practice corresponding to values more than twice that of the next closest data point) were excluded from the analyses. Data from different chemicals were square root or natural log (ln) transformed as necessary to render them suitable for parametric analyses.
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Results |
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The detailed analysis of the phase change process, taking long-term gregarious locusts and then cohorts of insects isolated for periods of hours to three generations and then crowding third-generation solitarious locusts for periods of 4 h to one stadium, revealed significant changes in 12 of the 13 tested chemicals in at least one of the nine stages of isolation or crowding, (multivariate analysis of variance, MANOVA; Table 2, based on the total amounts present in the sampled regions of the central nervous system). Only N-acetyldopamine (Fig. 2M) showed no significant change with any of the treatments, or between regions of the central nervous system as either final instar nymphs (Table 2) or adults (Table 3). The data for final instar nymphs are divided into three patterns of response following isolation and crowding: amino acids that increased on isolation throughout the central nervous system (Figs 2AF, 3); chemicals that decreased on isolation throughout the central nervous system (Figs 2GI, 4) and the monoamines dopamine (Figs 2J, 5A), serotonin (Figs 2K, 5B) and octopamine (Figs 2L, 5C), which showed large regional changes, particularly during the early stages of isolation and crowding.
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Qualitatively, the differences between long-term gregarious adults and second-generation solitarious adults were similar to those of final instar nymphs for most chemicals (Table 3, Fig. 6). Aspartate, glutamate and glycine were present in approximately double the quantity in solitarious compared to gregarious adults, as in the final instar nymphs. GABA, arginine, taurine, dopamine and serotonin were also present in greater amounts in solitarious adults but had more extreme relative increases compared to nymphs, with solitarious adults also having approximately double the quantities of these chemicals compared to the 2035% increases seen for the same chemicals in final instar nymphs. There was less citrulline in gregarious adults but the difference was smaller than in nymphs (50% compared to >90% decrease, respectively), whilst there were no significant differences in the amounts of N-acetyldopamine, tyramine or octopamine. The acetylcholine analysis was not performed on the adult samples.
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Amino acids that increased on isolation
All the measured amino acids, except for citrulline, were present in
greater amounts throughout the central nervous systems in long-term
solitarious locusts than in long-term gregarious locusts (Figs
2AF,
3). Overall two patterns of
change with increasing isolation and then crowding could be seen
(Fig. 3A,B). One group,
consisting of aspartate, glutamate and glycine, showed large increases
(doubling or more) throughout the central nervous system following isolation
for a 24 h period (Fig. 3A; all
significant at P<0.05 in a Dunnet post hoc test against
the gregarious controls). Levels of all three chemicals, however, then fell
back towards gregarious values on isolation for an entire stadium
(Fig. 3A). Locusts that were
isolated for longer, i.e. 13 generations, had increasing amounts of
these amino acids with each generation of isolation, so that eventually the
amounts present after three generations were similar to the high levels
occurring after the initial 24 h period of isolation
(Fig. 3A).
Phase change in the opposite direction, gregarization produced by crowding third generation solitarious locusts for as little as 4 h, led to dramatic decreases (Fig. 3A) towards values found in long-term gregarious locusts. By 24 h of crowding, however, the amounts had increased again and were again similar to those of long-term isolated locusts. In the second group of chemicals, consisting of GABA, taurine and arginine, there were smaller (20%) or no increases following 24 h isolation of long-term gregarious locusts (all nonsignificant; Fig. 3B). On longer isolation, for one stadium, the mean amounts of all three amino acids even fell below those detected in long-term gregarious locusts.
For arginine and GABA, first-generation isolated locusts had increased amounts relative to locusts isolated for one stadium, but this was followed by only modest or no increases on each subsequent generation of isolation (Fig. 3B). Taurine differed somewhat from this pattern; there was a substantial increase (60%) relative to gregarious in the first and second generations of isolation but then a decrease to only 25% more than the gregarious level in the third generation of isolation (Fig. 3B). No other chemical showed the same change from the second to third generation of isolation. The amounts of arginine, taurine and GABA did not change when third-generation solitarious locusts were crowded for a 4 h period, but subsequently the amounts of taurine and GABA increased significantly after 24 h crowding.
Third-generation solitarious locusts that had been crowded for an entire stadium showed a strong decrease in the amounts of all amino acids in both groups towards gregarious values in the final larval instar (Fig. 3), and only the amounts of aspartate remained significantly different from those of long-term gregarious animals.
Chemicals that decreased on isolation of gregarious locusts
Three chemicals, citrulline, tyramine and acetylcholine, decreased
significantly following isolation of gregarious phase locusts,
(Fig. 4). The relative decrease
after three generations of isolation ranged from over 90% for citrulline, to
50% for tyramine and 20% for acetylcholine. Acetylcholine decreased
progressively with increasing periods of isolation, reaching a minimum on the
first generation of isolation and staying at a constant level thereafter. By
contrast there was a rapid decrease in citrulline and tyramine following
isolation for 24 h, but the amounts had increased somewhat (in the direction
of the gregarious amounts) after 1 stadium of isolation. They subsequently
decreased again during 13 generations of isolation. This pattern of
change shown by citrulline and tyramine
(Fig. 4) mirrored that of the
amino acids that increased on isolation, as described above.
Crowding third-generation solitarious locusts for 4 h to 1 stadium had no effect on the amount of acetylcholine, which remained consistently lower than in long-term gregarious locusts. Crowding third-generation solitarious locusts caused an increase in citrulline within 4 h, but by 24 h of crowding amounts had decreased again towards fully solitarious values. Crowding had no significant effect on the amount of tyramine after 4 h of crowding, but after crowding for 24 h there was a dramatic increase up to near gregarious levels, which was sustained after 1 stadium of crowding.
Dopamine
Dopamine increased three- to fivefold in all three regions of the central
nervous system following 24 h isolation of gregarious locusts
(Fig. 5A). This was followed by
a decline back towards gregarious values following 1 stadium of isolation, a
pattern similar to some of the amino acids (cf.
Fig. 3A). Isolation for
13 generations led to some increase in the amount of dopamine in the
brain and thoracic ganglia but measured amounts were highly variable between
samples and the differences were not significant in final instar nymphs. In
adults, however, crowding for two generations caused a significant elevation
of dopamine (Fig. 6,
Table 3). Amounts of dopamine
fell in the optic lobes and thoracic ganglia during 4 h of crowding of
third-generation solitarious nymphs to levels below those found in long-term
gregarious nymphs. Indeed, no dopamine was detected in any of the optic lobe
samples after 4 h of crowding (Fig.
5A). In the brain, dopamine levels after a 4 h period of crowding
remained similar to those of third-generation solitarious locusts,
approximately 2.5 times greater than that of long-term gregarious animals.
After 24 h of crowding, amounts of dopamine in the brain and optic lobes were
significantly greater than in long-term gregarious locusts, but after 1
stadium of crowding there was a decrease towards long-term gregarious values
in all three regions.
Serotonin
The mean amount of serotonin in the brain doubled and there was an
eightfold increase in the optic lobes following 24 h isolation of long-term
gregarious locusts (Fig. 5B).
This was followed by a decrease so that animals isolated for 1 stadium had
amounts of serotonin similar to those in long-term gregarious locusts. There
were increases in the mean amounts present in the brain and optic lobes of
one- to three-generation isolated locusts but, as with dopamine, these changes
were non-significant in nymphs whilst significant in second-generation
solitarious adults (Fig. 6,
Table 3). Isolation for 24 h to
three generations caused no change in the amount of serotonin present in the
thoracic ganglia. Crowding third-generation solitarious nymphs for 4 h led to
a small decrease in the amounts of serotonin found in the brain and optic
lobes, but to a ninefold increase in the thoracic ganglia. After 24 h crowding
there were fourfold increases in the brain and optic lobes, but amounts
present in the thoracic ganglia had declined again to near their gregarious
level. After 1 stadium of crowding, amounts of serotonin in all three regions
of the central nervous system were similar to those of long-term gregarious
locusts.
Octopamine
There were no statistically significant changes in the amount of octopamine
present throughout the central nervous system during isolation (from 24 h to
three generations). There was, however, considerable variation in the amounts
detected in the optic lobes and thoracic ganglia of locusts isolated for 24 h
to two generations, with the mean amounts well above those of long-term
gregarious nymphs (Fig. 5C).
There was no significant difference between amounts detected in long-term
gregarious and second generation solitarious adult locusts
(Fig. 6). Crowding long-term
solitarious locusts, however, produced large changes in the amount of
octopamine. Crowding for 4 h caused octopamine to decrease to an undetectable
level throughout the central nervous system
(Fig. 5C), paralleling changes
seen in dopamine levels in the optic lobes and thoracic ganglia
(Fig. 5A). After 24 h of
crowding, however, octopamine levels increased by a mean of eightfold in the
thoracic ganglia, and by a mean of 14-fold in the optic lobes, although there
was great variability between samples. After 1 stadium of crowding, amounts of
octopamine present in the thoracic ganglia and brain were near those of
gregarious locusts, but the amount present in the optic lobes remained 15
times larger than in long-term gregarious locusts.
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Discussion |
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Long-term differences
The different patterns of changes amongst the sampled chemicals argue
against phase change causing a single overarching adjustment in metabolic
rate, or even overall growth of the central nervous system. During isolation
relative increases in chemicals ranged from 30% to over 300% for the amino
acids, and some chemicals decreased in the same period. There were no simple
correlations between changes in the amount of one chemical and changes in a
related chemical compound. Thus, for example, although GABA is synthesised
from glutamate, the observed doubling of glutamate in solitarious locusts was
accompanied by only a 30% increase in GABA. Tyramine is a precursor of
octopamine and is possibly a neuromodulator in its own right
(Downer et al., 1993;
Roeder et al., 2003
) but,
whilst amounts of tyramine decreased in long-term solitarious locusts, amounts
of octopamine were no different between phases. NADA is a metabolite of
dopamine (Sasaki and Nagao, 2000), but whilst amounts of dopamine differed
between phases, those of NADA did not.
The monoamines analysed play important roles in the regulation and
modulation of insect nervous systems and may control or modify many
physiological processes and behaviours
(Burrows, 1996;
Homberg, 2002
). Only a small
number of neurones in the fully gregarious locust show antibody staining to
serotonin, but many of these neurones have extensive ramifications throughout
the central nervous system, including most of the major neuropiles of the
brain (Klemm and Sundler,
1983
; Homberg,
1991
). In each of the thoracic ganglia there are two large and up
to three small pairs of cell bodies associated with each neuromere
(Tyrer et al., 1984
). There
are about 3000 neurones showing dopamine-like immunoreactivity in the
peripheral optic lobes of locusts, with a further 110 pairs of
dopamine-containing neurones in the central part of the brain, with notable
absences of staining in the mushroom bodies, olfactory regions of the antennal
lobes and most of the lobula (Wendt and
Homberg, 1992
). In the thoracic ganglia, there are three pairs of
cells showing dopamine-like immunoreactivity in the prothoracic ganglion, one
pair in the mesothoracic and none in the metathoracic. Serotoninergic,
dopaminergic and octopaminergic fibres all supply the storage lobes of the
corpora cardiaca (Konings et al.,
1988
).
By contrast with the monoamines, the roles of some of the amino acids are largely unknown. Although absolute amounts of amino acids detected differed considerably in different regions, the relative changes they underwent during phase change were generally consistent throughout the whole of the central nervous system, suggesting that there are some universal changes in neuronal function, in addition to specific modification of key neuronal networks.
Aspartate, glutamate, glycine and arginine are all structural amino acid
monomers used in protein synthesis and would therefore be expected to be found
in any tissue. The rapidity of the changes in the amounts of three of these
amino acids in locusts subjected to short-term crowding or isolation argues
against the differences being explained by changes in the relative size of the
brain between phases. Nor can the differences be explained by changes in
dietary protein intake, which remains unchanged between phases in locusts fed
a nutritionally optimal diet, as in the present study
(Simpson et al., 2002). An
additional set of samples taken from another body tissue might suggest whether
the changes in the amounts of structural amino acids are a general consequence
of phase change or more specific to the central nervous system. The fat body,
however, differs in extent and composition between phases
(Ayali et al., 1996
;
Pener et al., 1997
),
suggesting that different organs may undergo their own characteristic
modifications during phase change. By contrast, the muscles are innervated by
neurones using many of the same substances that we have analysed, and
therefore differences are likely to reflect those found in the central nervous
system.
Glutamate, GABA and acetylcholine are widespread and important
neurotransmitters in the central and peripheral nervous systems of insects.
Glutamate is thought to be the principal excitatory transmitter at
neuromuscular junctions, and is therefore strongly associated with motor
neurones, but it also acts as a central neurotransmitter
(Wafford and Sattelle, 1989;
Parker, 1994
). There are
350600 cell bodies displaying strong glutamate immunoreactivity in each
of the thoracic ganglia (Watson and
Seymour-Laurent, 1993
). The inhibitory neurotransmitter GABA has a
wide distribution in the central nervous system, as demonstrated by
immunocytochemistry (Breer and Heilgenberg,
1985
). In locust thoracic ganglia, each ganglion has approximately
250 cell bodies exhibiting GABA-like immunoreactivity including inhibitory
motor neurones, a population of spiking local interneurones
(Watson, 1986
) and non-spiking
local interneurones (Wildman et al.,
2002
). Acetylcholine is thought to be an important excitatory
neurotransmitter of locust mechanosensory
(Lutz and Tyrer, 1988
;
Parker and Newland, 1995
;
Gauglitz and Pflüger,
2001
) and chemosensory afferents
(Python and Stocker, 2002
).
The decrease in the amount of acetylcholine on increasing solitarization would
be congruent with the observed decrease in the numbers of exteroceptive and
chemoreceptive sensilla, and therefore sensory afferents, on most leg segments
in solitarious locusts (Rogers et al.,
2003
).
Arginine is the precursor of nitric oxide (NO) and citrulline is a
metabolic byproduct of NO formation
(Palmer et al., 1988).
Citrulline is produced in stoichiometric amounts to NO and has been shown
previously to be a reliable index of NO release
(Kendrick et al., 1996
).
Whereas the differences in the amounts of arginine could arise from a number
of causes, the reciprocal changes in the amounts of citrulline on increasing
solitarization provide an indication, albeit indirectly, that NO signalling
differs between phases throughout the central nervous system. NO is a rapidly
diffusing intercellular signalling molecule synthesised by many neurones in
the locust central nervous system (Ott et
al., 2001
). These include neuronal populations in the optic lobes
(Elphick et al., 1996), the antennal lobes, which show the highest
concentration of nitric oxide synthase in the locust brain
(Müller and Bicker, 1994
)
and mechanosensory processing neuropiles in the thoracic ganglia
(Ott and Burrows, 1998
).
The remaining amino acids, glycine, aspartate and taurine, have as yet
largely undefined roles in the central nervous system of insects. Glycine is
an important inhibitory neurotransmitter in vertebrates
(Kuhse et al., 1995), but has
no known neuronal function in invertebrates. Despite this, glycine showed some
of the strongest differences during phase change, paralleling the pattern of
change seen in glutamate and aspartate. Immunocytochemical staining has
revealed regions of strong aspartate staining in the lamina of the optic lobe
that are widely conserved in phylogenetically distant groups of insects
(Sinakevitch and Strausfeld,
2004
). There are distinct aspartate-rich laminas in the mushroom
bodies of bees and cockroaches that alternate with taurine-rich regions
(Ehmer and Gronenberg, 2002
;
Sinakevitch et al., 2001
).
Aspartate is also an agonist of one class of cation-selective glutamate
receptor (Usherwood, 1994
) and
may therefore have a functional role in signalling of some glutaminergic
neurones. Taurine is abundant in the central nervous system of insects
(Schafer et al., 1988
) and the
mushroom bodies contain discrete laminas that stain strongly for taurine,
hinting at specific functional roles
(Sinakevitch and Strausfeld,
2004
).
Rapid changes on crowding or isolation
Behavioural gregarization is fully established within 4 h of crowding
(Roessingh and Simpson, 1994;
Fig. 6B), and over this period
locusts shift from being repelled by other locusts to being strongly attracted
(Simpson et al., 1999
).
Subsequently, other locusts provide gregarizing stimuli, continuously
reinforcing the gregarious phase state in a positive feedback loop. Therefore,
the changes that occurred within just hours of crowding third-generation
solitarious locusts might have a particular functional significance in the
initiation of phase change. Eight of the twelve chemicals that showed
significant differences with phase state dropped towards or even below
gregarious values with 4 h crowding of third-generation solitarious locusts
(Fig. 6B). The amount of
thoracic serotonin, however, stood out from this trend in that it increased
ninefold during the first 4 h of crowding. This massive change in serotonin in
just the thoracic ganglia may be particularly significant to the early stages
of gregarization, as only sensory receptors on the middle and particularly the
hind legs can detect key mechanosensory gregarizing stimuli
(Simpson et al., 2001
;
Rogers et al., 2003
).
The rapid chemical changes that occurred on initial crowding were not
sustained; after crowding for a 24 h period, the amounts of most amino acids
were more similar to those of long-term solitarious locusts than those that
had been crowded for just 4 h. Amounts of serotonin (in the brain only),
dopamine and octopamine (optic lobes and thoracic ganglia), however, having
decreased in the first 4 h of crowding, subsequently increased above the
levels of long-term solitarious locusts during this period. Data from
Roessingh and Simpson (1994)
suggest that there is a small degree of reversion to a more solitarious
behavioural state in animals crowded for 24 h relative to their values after 4
h (Fig. 6B). This was possibly
due to the fact that 12 h of the crowding period was spent in the dark, during
which time activity levels, and thus interactions among individuals, would
have been greatly reduced. This could also account for some of the reversion
to solitarious levels of neurochemicals, but the degree of neurochemical
change is more extreme than that suggested by the behavioural reversion.
Locusts crowded for a longer period, the final larval stadium, showed a strong shift in levels of most chemicals towards amounts characteristic of long-term gregarious locusts, with octopamine in the optic lobes, acetylcholine and citrulline being the notable exceptions. These changes largely concur with the robust behavioural gregarization shown by locusts after this time.
The expression of solitarious behaviour follows a different pattern from
that of gregarization. In locusts that have been gregarious for generations
there is an initial, rapid phase of solitarization over the first 4 h of
isolation but this stabilizes at an intermediate level of solitarious
behaviour [median P (solitary)0.4;
Roessingh and Simpson, 1994
;
Simpson et al., 1999
;
Fig. 6B]. Locusts can only
become more solitarious if kept isolated across stadia and generations. By
contrast, when locusts that have been solitarious for generations are briefly
crowded for 2496 h they become fully behaviourally gregarious. When
reisolated, they return to a fully solitarious state within a few hours
(Roessingh and Simpson, 1994
).
This suggests that solitarization is at least a two-stage process.
The rapid change in the amounts of many neurochemicals that occurred during the initial (24 h) isolation period of gregarious locusts has a partial correlate in the rapid partial behavioural solitarization that occurs over this period (Fig. 6B). Many chemicals, however, increased to levels equal to those of long-term solitarious locusts, and thus exceeded the equivalent behavioural change. Amounts of eight of the twelve chemicals were either more than doubled or halved in quantity within 24 h of isolating gregarious locusts. Changes that occurred in dopamine and levels of serotonin in the brain and particularly optic lobes in the first 24 h of isolation exceeded those of long-term solitarious locusts. Large changes in serotonin are therefore implicated in both the early stages of gregarization and solitarization, but occur in a different location within the central nervous system, i.e. the thoracic ganglia during gregarization and the brain during solitarization. As with the effect of crowding, these large initial changes were not sustained in the medium term and amounts of most chemicals found in locusts isolated for one stadium were more similar to those in long-term gregarious locusts than to those in animals that had been isolated for just 24 h, and it required a generation or more of isolation for more stable chemical differences to appear.
Only octopamine has previously been measured with regard to phase change
and the results were unclear. Morton and Evans
(1983) found no phase-related
difference in octopamine in Schistocerca americana gregaria, whereas
Fuzeau-Braesch and colleagues
(Fuzeau-Braesch and David,
1978
; Fuzeau-Braesch and
Nicholas, 1981
), using Locusta migratoria, suggested
there were strong differences, with solitarious phase locusts containing more
octopamine than gregarious insects. It should be noted, however, that although
S. americana changes its colour with rearing density, behavioural
phase change is much weaker in this species
(Sword, 2003
). We found no
differences between amounts of octopamine in long-term solitarious and
gregarious desert locusts, but the large increase we saw after the initial
stages of gregarization (when levels fell) may account for the data obtained
by Fuzeau-Braesch and colleagues if their husbandry or handling procedures
inadvertently led to behavioural gregarization of their solitarious
animals.
Octopamine is often loosely characterised as a `stress hormone'
(Davenport and Evans, 1984a;
Birmingham and Tauck, 2003
),
and levels have been shown to increase in the haemolymph by up to eightfold in
response to conditions such as food deprivation
(Davenport and Evans, 1984b
)
or heat stress (Hirashima et al.,
2000
). Indeed, the widespread changes in brain chemicals we report
here, whilst clearly correlated with both solitarization and gregarization, do
not necessarily have a causative role. These rapid changes may instead arise
from an aversive response to being subjected to a novel situation, i.e. being
placed in or out of a group unexpectedly, and should perhaps be seen as
another kind of phase characteristic.
We have previously demonstrated specific neurophysiological differences
between the two extreme locust phases
(Matheson et al., 2004), which
may in part be underpinned by differences in synaptic transmission and
neuromodulation, and this will become a focus of future analyses at the
cellular and network level. Previous immunocytochemical analyses of neurones
containing the neurotransmitters and neuromodulators analysed in this study
have been performed only on long-term gregarious locusts. Perhaps phase change
may alter the branching patterns and numbers of neurones expressing different
neurochemicals, and this will be pursued in future studies. Our data
demonstrate the extensive neurochemical changes in the central nervous system
that accompany phase change in locusts and lay the foundation for more
critical examinations of the causative role these substances may have in
either gregarization or solitarization.
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List of abbreviations |
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Acknowledgments |
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Footnotes |
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Present address: Department of Biology, University of Leicester, University
Road, Leicester LE1 7RH, UK
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References |
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