Behavioural phase polyphenism in the Australian plague locust (Chortoicetes terminifera)

Abstract

Swarming and the expression of phase polyphenism are defining characteristics of locust species. Increases in local population density mediate morphological, physiological and behavioural changes within individuals, which correlate with mass marching of juveniles in migratory bands and flying swarms of adults. The Australian plague locust (Chortoicetes terminifera) regularly forms migratory bands and swarms, but is claimed not to express phase polyphenism and has accordingly been used to argue against a central role for phase change in locust swarming. We demonstrate that juvenile C. terminifera express extreme density-dependent behavioural phase polyphenism. Isolated-reared juveniles are sedentary and repelled by conspecifics, whereas crowd-reared individuals are highly active and are attracted to conspecifics. In contrast to other major locust species, however, behavioural phase change does not accumulate across generations, but shifts completely within an individual's lifetime in response to a change in population density.

Full text

Biol. Lett. (2009) 5, 306–309
doi:10.1098/rsbl.2008.0764
Published online 4 March 2009
Animal behaviour
Behavioural phase
polyphenism in the
Australian plague locust
(Chortoicetes terminifera)
Lindsey J. Gray, Gregory A. Sword*,
Michael L. Anstey, Fiona J. Clissold
and Stephen J. Simpson
School of Biological Sciences, The University of Sydney, Sydney,
New South Wales 2006, Australia
*Author for correspondence (greg.sword@bio.usyd.edu.au).
Swarming and the expression of phase polyphen-
ism are defining characteristics of locust species.
Increases in local population density mediate
morphological, physiological and behavioural
changes within individuals, which correlate with
mass marching of juveniles in migratory bands
and flying swarms of adults. The Australian
plague locust (Chortoicetes terminifera) regularly
forms migratory bands and swarms, but is
claimed not to express phase polyphenism and
has accordingly been used to argue against a
central role for phase change in locust swarming.
We demonstrate that juvenile C. terminifera
express extreme density-dependent behavioural
phase polyphenism. Isolated-reared juveniles are
sedentary and repelled by conspecifics, whereas
crowd-reared individuals are highly active and
are attracted to conspecifics. In contrast to
other major locust species, however, behavioural
phase change does not accumulate across
generations, but shifts completely within an
individual’s lifetime in response to a change in
population density.
Keywords: phenotypic plasticity; behaviour;
migration; phase polyphenism; insect
1. INTRODUCTION
At low population densities locusts express ‘solitarious’
phase characteristics, whereas high population densities
induce the expression of ‘gregarious’ phase phenotypes
from the same genotype. Although phase change
typically involves many different morphological and
physiological traits (Simpson & Sword 2008,2009;
Pener & Simpson in press), the change in behaviour
that occurs in response to crowding is considered to be
the principal driver underlying group formation and
mass movement by marching bands of juveniles
and flying swarms of adults (Simpson et al.1999;
Simpson & Sword 2009). Solitarious individuals are
relatively sedentary and repelled by conspecifics, but
high population densities induce individuals to become
more active and attracted to one another. The spatial
distribution of resources in locust habitats has been
shown to be critical in initially bringing solitarious
phase locusts together and promoting phase change
(Babah & Sword 2004), while the ensuing directional
mass movement of gregarious locusts at high popu-
lation densities is collectively determined and influ-
enced by cannibalistic interactions among individuals
(Bazazi et al.2008).
The Australian plague locust, Chortoicetes terminifera
(Acrididae: Oedipodinae) frequently outbreaks and
invades agricultural areas (Hunter 2004). Although
C. terminifera forms characteristic migratory bands and
swarms, it appears not to express density-dependent
changes in colour or morphology as seen in other
major locust species (Uvarov 1977). Accordingly,
C. terminifera is widely assumed not to express phase
polyphenism (Hunter 2004), an assertion that has
been used to question the importance of phase
polyphenism in locust swarm formation (Key 1950).
However, early field observations of C. terminifera
juvenile behaviour clearly described what appears to be
behavioural phase change (Clark 1949).
Here, we quantify behavioural phase polyphenism
in C. terminifera and show that it expresses density-
dependent behavioural changes very similar to those
of other major swarming locust species.
2. MATERIAL AND METHODS
Chortoicetes terminifera rearing and behavioural analysis were
modified from protocols for the desert locust, Schistocerca gregaria
(Roessingh et al. 1993,Simpson et al. 1999; see the electronic
supplementary material). Field-collected locusts were reared under
crowded conditions for multiple generations. From these, individ-
uals were reared in isolation for one, two and three generations.
To test for the expression of behavioural phase change, the
behaviour of individual mid-final instar nymphs reared either
continuously crowded or isolated for three generations was assayed,
using an automated video-tracking behavioural assay designed to
quantify individual locomotory and position-related responses to a
stimulus group of conspecifics.
Behavioural differences between isolated- and crowd-reared
locusts were compared using binar y logistic regression (BLR)
modelling (see the electronic supplementary material). The result-
ing logistic regression model, which successfully discriminated
between solitary-reared (solitarious) and crowd-reared (gregarious)
locusts, was used to calculate the probability of solitarious phase
group membership (P
solitarious
), providing a quantitative measure of
individual behavioural phase state for use as a dependent variable
in subsequent experiments.
The presence of transgenerational epigenetic transfer of phase
state, as known in other locust species (Miller et al.2008), was
assessed using group and pairwise comparisons of the P
solitarious
values of fifth-instar nymphs from cohorts reared in isolation for one,
two and three generations, and those from the continuously crowded
gregarious culture (see the electronic supplementary material).
To test for an effect of isolation across multiple nymphal stages
on the loss of gregarious behaviour, referred to as behavioural
solitarization, crowd-reared third- and fourth-instar nymphs were
isolated and assayed approximately one week later in the final
(fifth) nymphal instar. P
solitarious
scores of locusts removed from the
crowd were compared with those of continuously crowded fifth-
instar nymphs. Solitarization due to shorter term isolation within a
single instar was tested by compar ing the P
solitarious
values of
continuously crowded fifth-instar nymphs (24 hours post-ecdysis)
with those of similar locusts isolated for 72 hours (see the electronic
supplementary material).
To test for an effect of crowding across multiple nymphal stadia
on behavioural gregarization, second-generation solitary-reared
third- and fourth-instar locusts were crowded until assayed approxi-
mately one week later in the fifth instar. P
solitarious
values of the
recently crowded locusts were compared with those of continuously
isolated fifth-instar controls. Gregarization due to crowding within a
single instar was tested by comparing the P
solitarious
values of second-
generation solitary-reared fifth instars with those of similar locusts
crowded for 72 hours (see the electronic supplementary material).
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rsbl.2008.0764 or via http://rsbl.royalsocietypublishing.org.
Received 11 December 2008
Accepted 5 February 2009 306 This journal is q2009 The Royal Society

3. RESULTS
The behaviour of crowd-reared C. terminifera nymphs
differed significantly from that of solitary-reared
individuals. A forward-conditional stepwise BLR
model classified 100 per cent of solitary-reared and
97.4 per cent of crowd-reared locusts into their
correct rearing category, with an overall accuracy of
98.7 per cent (table 1). Crowd-reared nymphs moved
more, spent more time climbing and spent more time
near the stimulus group than did solitary-reared
individuals (table 1;figure 1).
Isolation of gregarious phase insects for a single
generation resulted in a complete loss of gregarious
behaviour, with no evidence of epigenetic transmission
across generations (Kruskal–Wallis test, KZ30.76,
p!0.0001, d.f.Z3). Pairwise tests were significant
between the long-term crowd-reared group and all
three solitary-reared groups with no differences between
first-, second- and third-generation solitary-reared
groups (Mann–Whitney U-test, all p!0.05; figure 2).
Within a single generation, long-term crowded
individuals shifted rapidly to the solitarious behavioural
Table 1. Behavioural variable coefficients retained in the most parsimonious forward-conditional logistic regression model
derived from 78 crowd-reared and 78 third-generation isolated-reared fifth-instar C. terminifera locusts. (Negative coefficients
indicate that the magnitude of response was greater in crowd-reared (gregarious) as opposed to solitary-reared (solitarious)
individuals. The Wald statistic indicates the significance of variable contributions to the model. The Hosmer and
Lemeshow goodness-of-fit test was not significant ( pZ0.859, d.f.Z8). (Model: hZb
0
Cb
1
X
1
Cb
2
X
2
C/Cb
k
X
k
, with
P
solitarious
Ze
h
/(1Ce
h
).))
variable coefficient bcoefficient b, s.e. Wald statistic
significance of
Wald statistic
average distance to the
stimulus chamber
0.438 0.155 7.956 0.005
distance moved K0.054 0.019 8.147 0.004
climb time K0.074 0.27 7.523 0.006
constant K0.359 1.32 0.074 0.785
1400
1200
1000
800
600
400
200
0
total distance moved (cm)
isolated crowded
(c)
25
20
15
10
5
0
average distance to the stimulus
chamber (cm)
400
200
0
total time climbing (s)
isolated crowdedisolated crowded
(a)(b)
Figure 1. Box plots representing (a–c) the behavioural variables retained in the logistic regression model from 78 crowd-
reared and 78 third-generation isolated-reared C. terminifera fifth-instar nymphs. Each box displays the median value, with
the ends of boxes representing the 25th and 75th percentiles and the ends of the whiskers representing the 10th and 90th
percentiles. Circles, outliers; asterisks, extreme outliers.
Phase polyphenism in locusts L. J. Gray et al. 307
Biol. Lett. (2009)

condition upon isolation. The P
solitarious
values of
crowd-reared fifth-instar nymphs isolated from the
third or fourth instar were significantly higher than
those in the non-isolated control group (UZ19,
pZ0.020; figure 2). Isolating crowd-reared nymphs for
72 hours resulted in the expression of solitarious phase
behaviour, with a median P
solitarious
value of 0.95 for
the treatment group versus 0 for the controls (UZ16,
pZ0.003; figure 2).
Crowding across multiple instars induced behavi-
oural phase change in solitary-reared locusts. The
P
solitarious
values of solitary-reared fifth-instar locusts
that had been crowded since either the third or fourth
nymphal stadium were significantly lower than those of
uncrowded controls (UZ30, pZ0.001; figure 2).
Behavioural gregarization was induced by crowding for
72 hours, with significantly lower P
solitarious
values
among locusts in the crowded treatment group relative
to the isolated controls (UZ17, pZ0.022; figure 2).
4. DISCUSSION
The Australian plague locust expresses classic
density-dependent behavioural phase polyphenism,
which is both qualitatively and quantitatively similar
to that reported in other major swarming locust
species (Simpson et al.1999;Pener & Simpson
in press). Nymphs reared under isolated conditions
were much more sedentary and repelled by conspe-
cifics relative to those reared under crowded con-
ditions, which were more active and attracted to other
locusts (figure 1).
The lack of obvious density-dependent phenotypic
changes in colour and morphology in C. terminifera
fostered the notion that phase polyphenism might not
play a role in the formation and mass movement of
locusts in migratory bands and swarms during out-
breaks (Key 1950;Hunter 2004). On the contrary,
our results for C. terminifera are consistent with
the predicted role of behavioural phase change as
a driver of locust swarm formation and mass move-
ment during outbreaks (Simpson & Sword 2009).
Importantly, the elucidation of behavioural phase
change in C. terminifera will enable the established
understanding of the relationship between locust
resource distribution patterns, gregarization and
swarm formation to be applied to improve locust
forecasting and management based on local habitat
information (Babah & Sword 2004).
Although the behaviour of solitarious and gregar-
ious phase C. terminifera nymphs is similar to that in
other locusts, the time course of its expression within
and across generations differs. Epigenetic trans-
mission of phase traits has been found in other locust
species, mediated by maternally produced gregarizing
chemicals (Miller et al. 2008). In the desert locust,
S. gregaria, whereas behavioural gregarization occurs
within hours, three successive generations of isolated
rearing are required before long-term crowded insects
express fully solitarious behaviour ( Roessingh et al.
1993). In C. terminifera, behavioural phase change
was complete in either direction within days (figure 2).
This result does not prove the absence of maternal
inheritance of phase in C. terminifera, rather it shows
solitarious 1.0
gregarious
0.2
0
0.4
0.6
0.8
isolated 72 h
isolated two generations
isolated one generation
isolated one week
isolated three generations (model)
crowded multiple generations
(model)
crowded 72 h
crowded one week
crowded multiple generations
solitarization gregarization
P
solitarious value
n = 78 22 22 11 10 14 10 22 78
Figure 2. Summary of the time course of behavioural gregarization and solitarization in C. terminifera. Filled circles represent
median (G95% CI) P
solitarious
values of final instar juveniles from the isolated and crowded treatment groups of different
durations. Open circles depict the P
solitarious
values of the long-term crowded and third-generation isolated locusts used to
construct the logistic regression model (table 1). Note that both gregarization and solitarization are achieved after 72 hours,
and that the behavioural state attained after 72 hours is equivalent to that attained after many generations of either crowded
or isolated rearing. Confidence intervals were obtained using the Resampling Stats add-in for EXCEL (Resampling Stats,
Inc. 2006).
308 L. J. Gray et al. Phase polyphenism in locusts
Biol. Lett. (2009)

that behavioural solitarization and gregarization pro-
ceed to completion so rapidly that behavioural phase
state does not accumulate over successive generations.
Perhaps this rapid time course of solitarization reflects
ecological differences between locust species in the
autocorrelation of local population densities across
generations, or, possibly, C. terminifera lacks genetic
variation at loci critical for the transmission of
epigenetic effects (e.g. Kucharski et al. 2008).
Locust swarming and the expression of phase
polyphenism appear to have arisen independently
numerous times, which gives rise to two questions
(Song 2005;Lovejoy et al. 2006;Simpson & Sword
2009): (i) what is the role of genetics versus the
environment in locust swarming and (ii) have unique
mechanisms underlying the expression of phase poly-
phenism independently evolved several times, or are
the differences among species due to modifications
of the same gene regulatory pathways? Functional
genomics resources for Locusta migratoria (Kang et al.
2004;Ma et al. 2006) offer tools for unravelling the
molecular genetic mechanisms underlying locust
phase change. Locusta migratoria and C. terminifera
are in the same subfamily (Oedipodinae), but
L. migratoria expresses extreme phase changes in
multiple traits including coloration and morphology,
whereas C. terminifera appears to change only in its
behaviour. Thus, comparative gene expression studies
hold potential for identifying the suite of genes
underlying behavioural phase change in these two
locusts. Similarities and differences between the
two will serve as a basis for broader phylogenetic
studies of the genetic and regulatory mechanisms
underlying locust phase change, thereby providing
insights into the evolution of phenotypic plasticity and
development of new pest management approaches.
This study was funded by Australian Research Council
Linkage Project grant LP0669080, in partnership with the
Australian Plague Locust Commission. We thank Laury
McCulloch, Martin Steinbauer, Tim Dodgson and Naz
Soran for their assistance.
Babah, M. A. O. & Sword, G. A. 2004 Linking locust
gregarization to local resource distribution patterns
across a large spatial scale. Environ. Entomol. 33,
1577–1583.
Bazazi, S., Buhl, J., Hale, J. J., Anstey, M. L., Sword,
G. A., Simpson, S. J. & Couzin, I. D. 2008 Collective
motion and cannibalism in locust migratory bands. Curr.
Biol. 18, 735–739. (doi:10.1016/j.cub.2008.04.035)
Clark, L. R. 1949 Behaviour of swarm hoppers of the
Australian plague locust (Chortoicetes terminifera Walker).
CSIRO Bull. 245, 1–27.
Hunter, D. M. 2004 Advances in the control of locusts
(Orthoptera: Acrididae) in eastern Australia: from crop
protection to preventative control. Aust. J. Entomol. 43,
293–303. (doi:10.1111/j.1326-6756.2004.00433.x)
Kang, L., Chen, X., Zhou, Y., Liu, B., Zheng, W., Li, R.,
Wang, J. & Yu, J. 2004 The analysis of large-scale gene
expression correlated to the phase changes of the
migratory locust. Proc. Natl Acad. Sci. USA 101,
17 611–17 615. (doi:10.1073/pnas.0407753101)
Key, K. H. L. 1950 A critique on the phase theory of
locusts. Q. Rev. Biol. 25, 363–407. (doi:10.1086/
397834)
Kucharski, R., Maleszka, J., Foret, S. & Maleszka, R. 2008
Nutritional control of reproductive status in honeybees
via DNA methylation. Science 319, 1827–1830. (doi:10.
1126/science.1153069)
Lovejoy, N. R., Mullen, S. P., Sword, G. A., Chapman,
R. F. & Harrison, R. G. 2006 Ancient trans-Atlantic
flight explains locust biogeography: molecular phylo-
genetics of Schistocerca.Proc. R. Soc. B 273, 767–774.
(doi:10.1098/rspb.2005.3381)
Ma, Z., Yu, J. & Kang, L. 2006 LocustDB: a relational
database for the transcriptome and biology of the
migratory locust (Locusta migratoria). BMC Genomics 7,11.
(doi:10.1186/1471-2164-7-11)
Miller, G. A., Islam, M. S., Claridge, T. D. W., Dodgson, T. &
Simpson, S. J. 2008 Swarm formation in the desert locust
Schistocerca gregaria: isolation and NMR analysis of the
primary maternal gregarizing agent. J. Ex p. Bio l. 211,
370–376. (doi:10.1242/jeb.013458)
Pener, M. P. & Simpson, S. J. In press. Locust phase
polyphenism: an update. Adv. Insect Physiol.
Roessingh, P., Simpson, S. J. & James, S. 1993 Analysis of
phase-related changes in behaviour of desert locust
nymphs. Proc. R. Soc. B 252, 43–49. (doi:10.1098/rspb.
1993.0044)
Simpson, S. J. & Sword, G. A. 2008 Locusts. Curr. Biol. 18,
364–366. (doi:10.1016/j.cub.2008.02.029)
Simpson, S. J. & Sword, G. A. 2009 Phase polyphenism in
locusts: mechanisms, population consequences, adaptive
significance and evolution. In Phenotypic plasticity of
insects: mechanisms and consequences (eds D. Whitman &
T. Ananthakrishnan), pp. 147–190. Plymouth, UK:
Science Publishers Inc.
Simpson, S. J., McCaffery, A. R. & Ha¨gele, B. F. 1999
A behavioral analysis of phase change in the desert
locust. Biol. Rev. 74, 461–480. (doi:10.1017/S0006
32319900540X)
Song, H. 2005 Phylogenetic perspectives on the evolution of
locust phase polyphenism. J. Orth opt. R es . 14, 235–245.
(doi:10.1665/1082-6467(2005)14[235:PPOTEO]2.0.CO;2)
Uvarov, B. P. 1977 Grasshoppers and locusts, vol. 2. London,
UK: Centre for Overseas Pest Research.
Phase polyphenism in locusts L. J. Gray et al. 309
Biol. Lett. (2009)