First draft genome assembly of the desert locust, Schistocerca gregaria.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust's use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.

Evaluation of linear and non-linear activation dynamics models for insect muscle.

In computational modelling of sensory-motor control, the dynamics of muscle contraction is an important determinant of movement timing and joint stiffness. This is particularly so in animals with many slow muscles, as is the case in insects-many of which are important models for sensory-motor control. A muscle model is generally used to transform motoneuronal input into muscle force. Although standard models exist for vertebrate muscle innervated by many motoneurons, there is no agreement on a parametric model for single motoneuron stimulation of invertebrate muscle. Although several different models have been proposed, they have never been evaluated using a common experimental data set. We evaluate five models for isometric force production of a well-studied model system: the locust hind leg tibial extensor muscle. The response of this muscle to motoneuron spikes is best modelled as a non-linear low-pass system. Linear first-order models can approximate isometric force time courses well at high spike rates, but they cannot account for appropriate force time courses at low spike rates. A linear third-order model performs better, but only non-linear models can account for frequency-dependent change of decay time and force potentiation at intermediate stimulus frequencies. Some of the differences among published models are due to differences among experimental data sets. We developed a comprehensive toolbox for modelling muscle activation dynamics, and optimised model parameters using one data set. The "Hatze-Zakotnik model" that emphasizes an accurate single-twitch time course and uses frequency-dependent modulation of the twitch for force potentiation performs best for the slow motoneuron. Frequency-dependent modulation of a single twitch works less well for the fast motoneuron. The non-linear "Wilson" model that optimises parameters to all data set parts simultaneously performs better here. Our open-access toolbox provides powerful tools for researchers to fit appropriate models to a range of insect muscles.

The Role of the Mechanotransduction Ion Channel Candidate Nanchung-Inactive in Auditory Transduction in an Insect Ear.

Insect auditory receivers provide an excellent comparative resource to understand general principles of auditory transduction, but analysis of the electrophysiological properties of the auditory neurons has been hampered by their tiny size and inaccessibility. Here we pioneer patch-clamp recordings from the auditory neurons of Müller's organ of the desert locust Schistocerca gregaria to characterize dendritic spikes, axonal spikes, and the transduction current. We demonstrate that dendritic spikes, elicited by sound stimuli, trigger axonal spikes, and that both types are sodium and voltage dependent and blocked by TTX. Spontaneous discrete depolarizations summate upon acoustic stimulation to produce a graded transduction potential that in turn elicits the dendritic spikes. The transduction current of Group III neurons of Müller's organ, which are broadly tuned to 3 kHz, is blocked by three ion channel blockers (FM1-43, streptomycin, and 2-APB) that are known to block mechanotransduction channels. We investigated the contribution of the candidate mechanotransduction ion channel Nanchung-Inactive-which is expressed in Müller's organ-to the transduction current. A specific agonist of Nanchung-Inactive, pymetrozine, eliminates the sound-evoked transduction current while inducing a tonic depolarizing current of comparable amplitude. The Nanchung-Inactive ion channels, therefore, have the required conductance to carry the entire transduction current, and sound stimulation appears not to open any additional channels. The application of three mechanotransduction ion channel blockers prevented the pymetrozine-induced depolarizing current. This implies that either Nanchung-Inactive is, or forms part of, the mechanotransduction ion channel or it amplifies a relatively small current (<30 pA) produced by another mechanotransduction ion channel such as NompC.SIGNIFICANCE STATEMENT The mechanically activated ion channel underpinning hearing is not known. We have pioneered intracellular patch-clamp recordings from locust auditory neurons to unravel the role of the candidate mechanotransduction ion channel Nanchung-Inactive in auditory transduction in insects.

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.

Solitary and gregarious locusts differ in circadian rhythmicity of a visual output neuron.

Locusts demonstrate remarkable phenotypic plasticity driven by changes in population density. This density dependent phase polyphenism is associated with many physiological, behavioral, and morphological changes, including observations that cryptic solitarious (solitary-reared) individuals start to fly at dusk, whereas gregarious (crowd-reared) individuals are day-active. We have recorded for 24-36 h, from an identified visual output neuron, the descending contralateral movement detector (DCMD) of Schistocerca gregaria in solitarious and gregarious animals. DCMD signals impending collision and participates in flight avoidance maneuvers. The strength of DCMD's response to looming stimuli, characterized by the number of evoked spikes and peak firing rate, varies approximately sinusoidally with a period close to 24 h under constant light in solitarious locusts. In gregarious individuals the 24-h pattern is more complex, being modified by secondary ultradian rhythms. DCMD's strongest responses occur around expected dusk in solitarious locusts but up to 6 h earlier in gregarious locusts, matching the times of day at which locusts of each type are most active. We thus demonstrate a neuronal correlate of a temporal shift in behavior that is observed in gregarious locusts. Our ability to alter the nature of a circadian rhythm by manipulating the rearing density of locusts under identical light-dark cycles may provide important tools to investigate further the mechanisms underlying diurnal rhythmicity.

A formal mathematical framework for physiological observations, experiments and analyses.

Experiments can be complex and produce large volumes of heterogeneous data, which make their execution, analysis, independent replication and meta-analysis difficult. We propose a mathematical model for experimentation and analysis in physiology that addresses these problems. We show that experiments can be composed from time-dependent quantities, and be expressed as purely mathematical equations. Our structure for representing physiological observations can carry information of any type and therefore provides a precise ontology for a wide range of observations. Our framework is concise, allowing entire experiments to be defined unambiguously in a few equations. In order to demonstrate that our approach can be implemented, we show the equations that we have used to run and analyse two non-trivial experiments describing visually stimulated neuronal responses and dynamic clamp of vertebrate neurons. Our ideas could provide a theoretical basis for developing new standards of data acquisition, analysis and communication in neurophysiology.

Motor neurone responses during a postural reflex in solitarious and gregarious desert locusts.

Desert locusts show extreme phenotypic plasticity and can change reversibly between two phases that differ radically in morphology, physiology and behaviour. Solitarious locusts are cryptic in appearance and behaviour, walking slowly with the body held close to the ground. Gregarious locusts are conspicuous in appearance and much more active, walking rapidly with the body held well above the ground. During walking, the excursion of the femoro-tibial (F-T) joint of the hind leg is smaller in solitarious locusts, and the joint is kept more flexed throughout an entire step. Under open loop conditions, the slow extensor tibiae (SETi) motor neurone of solitarious locusts shows strong tonic activity that increases at more extended F-T angles. SETi of gregarious locusts by contrast showed little tonic activity. Simulated flexion of the F-T joint elicits resistance reflexes in SETi in both phases, but regardless of the initial and final position of the leg, the spiking rate of SETi during these reflexes was twice as great in solitarious compared to gregarious locusts. This increased sensory-motor gain in the neuronal networks controlling postural reflexes in solitarious locusts may be linked to the occurrence of pronounced behavioural catalepsy in this phase similar to other cryptic insects such as stick insects.

Motor planning: insects do it on the hop.

Planning a motor action in advance of its possible execution doesn't require cortical complexity: a recent paper demonstrates that even fruit flies prepare for movement some time before they decide whether or not to take action.

Co-contraction and passive forces facilitate load compensation of aimed limb movements.

Vertebrates and arthropods are both capable of load compensation during aimed limb movements, such as reaching and grooming. We measured the kinematics and activity of individual motoneurons in loaded and unloaded leg movements in an insect. To evaluate the role of active and passive musculoskeletal properties in aiming and load compensation, we used a neuromechanical model of the femur-tibia joint that transformed measured extensor and flexor motoneuron spikes into joint kinematics. The model comprises three steps: first, an activation dynamics module that determines the time course of isometric force; second, a pair of antagonistic muscle models that determine the joint torque; and third, a forward dynamics simulation that calculates the movement of the limb. The muscles were modeled in five variants, differing in the presence or absence of force-length-velocity characteristics of the contractile element, a parallel passive elastic element, and passive joint damping. Each variant was optimized to yield the best simulation of measured behavior. Passive muscle force and viscous joint damping were sufficient and necessary to simulate the observed movements. Elastic or damping properties of the active contractile element could not replace passive elements. Passive elastic forces were similar in magnitude to active forces caused by muscle contraction, generating substantial joint stiffness. Antagonistic muscles co-contract, although there was no motoneuronal coactivation, because of slow dynamics of muscle activation. We quantified how co-contraction simplified load compensation by demonstrating that a small variation of the motoneuronal input caused a large change in joint torque.

A posture optimization algorithm for model-based motion capture of movement sequences.

We have developed and evaluated a new optical motion capture approach that is suitable for a wide range of studies in neuroethology and motor control. Based on the stochastic search algorithm of Simulated Annealing (SA), it utilizes a kinematic body model that includes joint angle constraints to reconstruct posture from an arbitrary number of views. Rather than tracking marker trajectories in time, the algorithm minimizes an error function that compares predicted model projections to the recorded views. Thus, each video-frame is analyzed independently from other frames, enabling the system to recover from incorrectly analyzed postures. The system works with standard computer and video equipment. Its accuracy is evaluated using videos of animated locust leg movements, recorded by two orthogonal views. The resulting joint angle RMS errors range between 0.7 degrees and 4.9 degrees, limited by the pixel resolution of the digital video. 3D-movement reconstruction is possible even from a single view. In a real experimental application, stick insect walking sequences are analyzed with leg joint angle deviations between 0.5 degrees and 3.0 degrees. This robust and accurate performance is reached in spite of marker fusions and occlusions, simply by exploiting the natural constraints imposed by a kinematic chain and a known experimental setup.

Load compensation in targeted limb movements of an insect.

The task of a multi-jointed limb making an aimed movement towards a target requires that the movement is regulated against external perturbations such as changing load. In particular, loading one part of a limb leads to altered static forces on all proximal segments, and to additional dynamic joint interaction forces when the limb moves. We have addressed the question of load compensation in an insect preparation in which a locust makes aimed scratching movements with a hind leg in response to tactile stimulation of a wing. We show that loading the femur or tibia with the equivalent of 8.5 times the mass of the tibia (corresponding to an increase of up to 11.6 times the rotational moment of inertia at the femur-tibia joint) does not impair the animal's ability to make well-coordinated, aimed movements of that leg towards different targets. The kinematics of the movements are the same, and animals aim the same part of their distal tibia at the target, regardless of loading. The movements are carried out with equal accuracy and at the same initial velocity under all load conditions. Because loading of the leg does not change the behavioural performance, there is no indication of a change in aiming strategy. This implies high leg joint stiffness and/or the existence of high gain proprioceptive control loops. We have previously shown that in the unloaded condition, movements elicited by stimuli to different places on the wing are driven by a single underlying movement pattern that shifts depending on stimulus location along the wing surface. Our present data show that leg proprioceptive inputs are also integrated into the leg motor networks, rendering hind limb targeting robust against large changes in moment of inertia.

Cell biology in the Antarctic: studying life in the freezer.

Many classical biologists working in Antarctica have 'gone molecular' to study the physiological basis for life on or below the ice. Investigating the remarkable adaptations of specific cells, organelles and molecules to this extreme environment can provide new perspectives on the processes studied in conventional experimental organisms.

Metathoracic neurons integrating intersegmental sensory information in the locust.

This paper describes the morphology and physiology of five types of local interneurons and three types of ascending intersegmental interneurons in the locust metathoracic ganglion that are points of convergence of sensory information from the wings. Four types of spiking local interneurons are members of a population with somata at the ventral midline. They are depolarised by stimulation of a metathoracic wing nerve, suggesting that they encode a sensory representation of this appendage. Some are also depolarised with short latencies following stimulation of a mesothoracic wing nerve, indicating that they collate intersegmental as well as local information. All the local interneurons have branches in the anterior ventral association centre or around the roots of the nerve that carries wing sensory neurons. This distinguishes them from other interneurons in the population. A fifth type of local interneuron that has unusual bilateral branching and is not a member of this population is described for the first time. The ascending interneurons are members of three populations. Neurons of each population have a characteristic pattern of responses to stimulation of the mesothoracic or metathoracic wing nerves, and some respond to tactile stimulation or movements of a hind leg. These latter interneurons thus collate information from both wings and legs. All three types of intersegmental interneurons have branches in the anterior ventral association centre or around the roots of the wing nerve. The responses of the interneurons described here shed new light on both local and intersegmental network function in this model system.