Darron A. Cullen

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

BackgroundThe Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.ResultsTaking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust.ConclusionArm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.

Rapid behavioural gregarization in the desert locust, Schistocerca gregaria entails synchronous changes in both activity and attraction to conspecifics.

Desert Locusts can change reversibly between solitarious and gregarious phases, which differ considerably in behaviour, morphology and physiology. The two phases show many behavioural differences including both overall levels of activity and the degree to which they are attracted or repulsed by conspecifics. Solitarious locusts perform infrequent bouts of locomotion characterised by a slow walking pace, groom infrequently and actively avoid other locusts. Gregarious locusts are highly active with a rapid walking pace, groom frequently and are attracted to conspecifics forming cohesive migratory bands as nymphs and/or flying swarms as adults. The sole factor driving the onset of gregarization is the presence of conspecifics. In several previous studies concerned with the mechanism underlying this transformation we have used an aggregate measure of behavioural phase state, Pgreg, derived from logistic regression analysis, which combines and weights several behavioural variables to characterise solitarious and gregarious behaviour. Using this approach we have analysed the time course of behavioural change, the stimuli that induce gregarization and the key role of serotonin in mediating the transformation. Following a recent critique that suggested that using Pgreg may confound changes in general activity with genuine gregarization we have performed a meta-analysis examining the time course of change in the individual behaviours that we use to generate Pgreg. We show that the forced crowding of solitarious locusts, tactile stimulation of the hind femora, and the short-term application of serotonin each induce concerted changes in not only locomotion-related variables but also grooming frequency and attraction to other locusts towards those characteristic of long-term gregarious locusts. This extensive meta-analysis supports and extends our previous conclusions that solitarious locusts undergo a rapid behavioural gregarization upon receiving appropriate stimulation for a few hours that is mediated by serotonin, at the end of which their behaviour is largely indistinguishable from locusts that have been in the gregarious phase their entire lives.

Behavioural phase change in the Australian plague locust, Chortoicetes terminifera, is triggered by tactile stimulation of the antennae

Density-dependent phase polyphenism is a defining characteristic of the paraphyletic group of acridid grasshoppers known as locusts. The cues and mechanisms associated with crowding that induce behavioural gregarization are best understood in the desert locust, Schistocerca gregaria, and involve a combination of sensory inputs from the head (visual and olfactory) and mechanostimulation of the hind legs, acting via a transient increase in serotonin in the thoracic ganglia. Since behavioural gregarization has apparently arisen independently multiple times within the Acrididae, the important question arises as to whether the same mechanisms have been recruited each time. Here we explored the roles of visual, olfactory and tactile stimulation in the induction of behavioural gregarization in the Australian plague locust, Chortoicetes terminifera. We show that the primary gregarizing input is tactile stimulation of the antennae, with no evidence for an effect of visual and olfactory stimulation or tactile stimulation of the hind legs. Our results show that convergent behavioural responses to crowding have evolved employing different sites of sensory input in the Australian plague locust and the desert locust.

Optimizing multivariate behavioural syndrome models in locusts using automated video tracking

Locusts exhibit a behavioural syndrome known as ‘behavioural phase polyphenism’, in which a number of behavioural traits change markedly in response to local population density. ‘Solitarious’ phase individuals, which are typical of low-density populations, change within hours from being relatively sedentary and repelled by other locusts to congregating actively with conspecifics and becoming more active (the ‘gregarious’ phase). In wild populations, this behavioural plasticity can lead to the emergence of mass marching bands of nymphs and winged adult swarms. Much of our understanding of behavioural phase transition comes from laboratory experiments, which routinely employ an arena-based assay to measure a suite of behavioural variables encompassing aspects of activity, movement pattern and responses towards a stimulus group of other locusts. Individuals are then quantitatively phenotyped along a linear scale from solitarious to gregarious, by entering their returned measurements for several behavioural characters into a logistic regression model. Recently, automated video tracking has enabled multiple experimenters to use a single behavioural model, rather than each having to construct their own. Here, we have taken advantage of another powerful feature of automated tracking systems: the opportunity to use stored data to conduct a rigorous optimization process, which both ensures that the derived statistical model encapsulates the multidimensional nature of locust behavioural phase to best effect, and also provides a new understanding of the relationship between different behaviours.

RNAi Unravels the Biology of the Hemimetabolous and Ametabolous Insects

Abstract RNA interference (RNAi) has proven to be among the most important developments in molecular biology-driven studies of insects over the last decade. Using exogenous double-stranded RNA to generate post-transcriptional knockdown phenotypes, this technique enables the functional characterization of an ever-increasing set of genetic sequence data. Whilst routinely employed in studies of model holometabolous insects like Drosophila melanogaster and Tribolium castaneum, RNAi has also facilitated research into the hemimetabolous and ametabolous orders. This looks set to increase with the recent publication of the first genomic datasets for insects without full metamorphosis; the pea aphid, Acyrthosiphon pisum, and the human body louse, Pediculus humanus humanus. In anticipation of a heightened interest in this field, this review surveys the diverse range of recent research employing RNAi in ametabolous and hemimetabolous insects and, in doing so, demonstrates the broad utility of the technique for future studies of development, physiology and behaviour.

Increased muscular volume and cuticular specialisations enhance jump velocity in solitarious compared with gregarious desert locusts, Schistocerca gregaria.

ABSTRACT The desert locust, Schistocerca gregaria, shows a strong phenotypic plasticity. It can develop, depending upon population density, into either a solitarious or gregarious phase that differs in many aspects of behaviour, physiology and morphology. Prominent amongst these differences is that solitarious locusts have proportionately longer hind femora than gregarious locusts. The hind femora contain the muscles and energy-storing cuticular structures that propel powerful jumps using a catapult-like mechanism. We show that solitarious locusts jump on average 23% faster and 27% further than gregarious locusts, and attribute this improved performance to three sources: first, a 17.5% increase in the relative volume of their hind femur, and hence muscle volume; second, a 24.3% decrease in the stiffness of the energy-storing semi-lunar processes of the distal femur; and third, a 4.5% decrease in the stiffness of the tendon of the extensor tibiae muscle. These differences mean that solitarious locusts can generate more power and store more energy in preparation for a jump than can gregarious locusts. This improved performance comes at a cost: solitarious locusts expend nearly twice the energy of gregarious locusts during a single jump and the muscular co-contraction that energises the cuticular springs takes twice as long. There is thus a trade-off between achieving maximum jump velocity in the solitarious phase against the ability to engage jumping rapidly and repeatedly in the gregarious phase. Highlighted Article: Larger muscles and more elastic cuticular springs allow solitarious locusts to jump 25% faster than gregarious locusts, but they require double the energy and time to operate the spring mechanism.

Take-off speed in jumping mantises depends on body size and a power-limited mechanism.

ABSTRACT Many insects such as fleas, froghoppers and grasshoppers use a catapult mechanism to jump, and a direct consequence of this is that their take-off velocities are independent of their mass. In contrast, insects such as mantises, caddis flies and bush crickets propel their jumps by direct muscle contractions. What constrains the jumping performance of insects that use this second mechanism? To answer this question, the jumping performance of the mantis Stagmomantis theophila was measured through all its developmental stages, from 5 mg first instar nymphs to 1200 mg adults. Older and heavier mantises have longer hind and middle legs and higher take-off velocities than younger and lighter mantises. The length of the propulsive hind and middle legs scaled approximately isometrically with body mass (exponent=0.29 and 0.32, respectively). The front legs, which do not contribute to propulsion, scaled with an exponent of 0.37. Take-off velocity increased with increasing body mass (exponent=0.12). Time to accelerate increased and maximum acceleration decreased, but the measured power that a given mass of jumping muscle produced remained constant throughout all stages. Mathematical models were used to distinguish between three possible limitations to the scaling relationships: first, an energy-limited model (which explains catapult jumpers); second, a power-limited model; and third, an acceleration-limited model. Only the model limited by muscle power explained the experimental data. Therefore, the two biomechanical mechanisms impose different limitations on jumping: those involving direct muscle contractions (mantises) are constrained by muscle power, whereas those involving catapult mechanisms are constrained by muscle energy. Summary: Praying mantises, which use direct muscle contractions to generate jumps, have take-off velocities limited by the amount of power (energy/time) the muscles can produce.