Muscle attachment sites of Phormia regina (Meigen).

An error was found in a previous publication on muscle attachment sites in forensically important blowfly larvae from 2012. The patterns we assigned to Lucilia illustris factually belong to Phormia regina.

Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain.

Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, we show that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.

Influence of food substrates on the development of the blowflies Calliphora vicina and Calliphora vomitoria (Diptera, Calliphoridae).

The blowflies Calliphora vicina and Calliphora vomitoria are among the first colonizers of human remains in Europe. Laboratory development studies with immature stages of these blowflies for postmortem interval (PMI) calculations are generally performed on different media such as processed food substrates or liver of various animals. The question arises whether these media per se influence the development of larvae and thus PMI calculations? In this systematic approach, the effects of an assortment of food substrates on the development of the larvae were analyzed. C. vomitoria showed much better growth on processed substrates such as beef, pork, turkey, and mixed minced meats than on unprocessed substrates such as beef and pork liver and turkey steak. Beef liver even impeded full development of the species and resulted in death of all individuals. C. vomitoria was therefore categorized as a specialist. Even though mixed minced meat yielded low pupariation rates for C. vicina, the species showed, otherwise, comparable growth rates on all substrates tested and was thus considered to be a generalist. These findings emphasize the importance of parameters besides temperature on the development rates of forensically important fly larvae.

Marked for life: muscle attachment site patterns in blowfly larvae are constant throughout development.

The muscular attachment sites (MAS) of blowfly larvae can be visualised as "dots" by removing and staining the cuticle. Each segment bears several rows of MAS. The silhouettes of a subset of those rows in the second, third, and fourth segments were previously shown to be specific for four species of L3 blowfly larvae. In this investigation, the MAS patterns are described for a fifth species (Protophormia terraenovae) and throughout larval development of Calliphora vicina and Calliphora vomitoria. The patterns of P. terraenovae show considerable differences to those of the Calliphora species (larger MAS, characteristic "M" shape in row 4A), thus providing further evidence for the viability of the method as tool for species determination. Larvae with a body length of only 3 mm already show a complete set of MAS expressing identical pattern characteristics as L3 larvae with maximal body length. These characteristics are largely unchanged throughout development. Plotting the row length as a function of the body length throughout development reveals a linear correlation. Therefore, in case of requirement (e.g. fragmentation), not only the species but also the approximate larval age can be calculated with this method.

Cuticular muscle attachment sites as a tool for species determination in blowfly larvae.

First results of a new method for species determination in third instar larvae of saprophagous blowflies are introduced. Cuticular attachment sites of a limited number of transversal muscles are visualized for light microscopic analysis. After removing the muscles and staining the cuticle, the attachment sites become visible as laterally symmetrical segmental clusters of dark dots. The combined patterns of five such clusters, located in the second, third and fourth segments, show sufficient differences to allow reliable separation of externally very similar larval Lucilia sericata and Lucilia illustris as well as Calliphora vomitoria and Calliphora vicina, the most common saprophagous blowfly species in Europe. Species determination even in poorly conserved, discoloured and fragmented blowfly larvae becomes possible with this new method. The method can primarily be applied for postmortem interval (PMI) calculations in forensic entomology. Interspecific morphological similarity of the larvae and differences in growth rate make species determination an essential requisite for an exact PMI calculation.

Gustatory feedback affects feeding related motor pattern generation in starved 3rd instar larvae of Calliphora vicina.

Gustatory feedback allows animals to distinguish between edible and noxious food and adapts centrally generated feeding motor patterns to environmental demands. In reduced preparations obtained from starved Calliphora larvae, putatively appetitive (ethanol), aversive (sodium acetate) and neutral (glucose) gustatory stimuli were applied to the anterior sense organs. The resulting sensory response was recorded from the maxillary and antennal nerves. All three stimuli increased the neural activity in both nerves. Recordings obtained from the antennal nerve to monitor the activation pattern of the cibarial dilator muscles, demonstrated an effect of gustatory input on the central pattern generator for feeding. Ethanol consistently enhanced the rhythmic activity of the CDM motor neurons either by speeding up the rhythm or by increasing the burst duration. Ethanol also had an enhancing effect on the motor patterns of a protractor muscle which moves the cephalopharyngeal skeleton relative to the body. Sodium acetate showed a state dependent effect: in preparations without spontaneous CDM activity it initiated rhythmic motor patterns, while an ongoing CDM rhythm was inhibited. Surprisingly glucose had an enhancing effect which was less pronounced than that of ethanol. Gustatory feedback therefore can modify and adapt the motor output of the multifunctional central pattern generator for feeding.

Simple clearing technique as species determination tool in blowfly larvae.

A simple clearing technique is presented by which species specific structures and organs of blowfly larvae can easily be visualized and displayed without any danger of mechanical damages or dislocations of delicate formations and without fixation of the object.

Feel the heat: The effect of temperature on development, behavior and central pattern generation in 3rd instar Calliphora vicina larvae.

Like in all poikilothermic animals, higher temperatures increase developmental rate and activity in Calliphora vicina larvae. We therefore could expect temperature to have a persistent effect on the output of the feeding and crawling central pattern generators (CPGs). When confronted with a steep temperature gradient, larvae show evasive behavior after touching the substrate with the cephalic sense organs. Beside this reflex behavior the terminal- and dorsal organ might also mediate long term CPG modulation. Both organs were thermally stimulated while their response was recorded from the maxillary- or antennal nerve. The terminal organ showed a tonic response characteristic while the dorsal organ was not sensitive to temperature. Thermal stimulation of the terminal organ did not affect the ongoing patterns of fictive feeding or crawling, recorded from the antennal- or abdominal nerve respectively. A selective increase of the central nervous system (CNS) temperature accelerated the motor patterns of both feeding and crawling. We propose that temperature affects centrally generated behavior via two pathways: short term changes like thermotaxis are mediated by the terminal organ, while long term adaptations like increased feeding rate are caused by temperature sensitive neurons in the CNS which were recently shown to exist in Drosophila larvae.

The thoracic muscular system and its innervation in third instar Calliphora vicina Larvae. I. Muscles of the pro- and mesothorax and the pharyngeal complex.

An anatomical description is given by the muscles in the pro- and mesothorax, and those associated with the feeding apparatus (cephalopharyngeal skeleton, CPS) that participate in feeding behavior in third instar Calliphora larvae. The body wall muscles in the pro- and mesothoracic segments are organized in three layers: internal, intermedial, and external. The muscles were labeled with roman numerals according to the nomenclature in use for the abdominal segments. Muscles associated with the CPS are labeled according to their function. The prothorax bears five pairs of lateral symmetrically longitudinal segmental body wall muscles and lacks the transversal muscle group present in the mesothorax and abdominal segments. Additionally, four pairs of intersegmental muscles project from the prothorax to the second, fourth, and fifth segment. The mesothorax bears 15 pairs of segmental longitudinal and 18 pairs of transversal muscles. The accessory pharyngeal muscles span the CPS and the cuticle. Three pairs of protractors and retractors and two pairs of mouth hook accessors (MH(AC)) exist, which move the CPS relative to the body. The pharyngeal muscles are exclusively attached to the structures of the CPS. The mouth hook elevators and depressors, which mediate the hooks rotation are attached to the ventral arm of the CPS and project to a dorsal (elevators) or ventral (depressors) protuberance of the mouth hooks. The cibarial dilator muscles (CDM) span the dorsal arms of the CPS and the dorsal surface of the esophagus and mediate food ingestion. The labial retractors (LRs) lack antagonists and project from the ventral surface of the CPS to the unpaired labium. Contractions of these muscles open the mouth cavity.

The thoracic muscular system and its innervation in third instar Calliphora vicina Larvae. II. Projection patterns of the nerves associated with the pro- and mesothorax and the pharyngeal complex.

We describe the anatomy of the nerves that project from the central nervous system (CNS) to the pro- and mesothoracic segments and the cephalopharyngeal skeleton (CPS) for third instar Calliphora larvae. Due to the complex branching pattern we introduce a nomenclature that labels side branches of first and second order. Two fine nerves that were not yet described are briefly introduced. One paired nerve projects to the ventral arms (VAs) of the CPS. The second, an unpaired nerve, projects to the ventral surface of the cibarial part of the esophagus (ES). Both nerves were tentatively labeled after the structures they innervate. The antennal nerve (AN) innervates the olfactory dorsal organ (DO). It contains motor pathways that project through the frontal connectives (FC) to the frontal nerve (FN) and innervate the cibarial dilator muscles (CDM) which mediate food ingestion. The maxillary nerve (MN) innervates the sensory terminal organ (TO), ventral organ (VO), and labial organ (LO) and comprises the motor pathways to the mouth hook (MH) elevator, MH depressor, and the labial retractor (LR) which opens the mouth cavity. An anastomosis of unknown function exists between the AN and MN. The prothoracic accessory nerve (PaN) innervates a dorsal protractor muscle of the CPS and sends side branches to the aorta and the bolwig organ (BO) (stemmata). In its further course, this nerve merges with the prothoracic nerve (PN). The architecture of the PN is extremely complex. It innervates a set of accessory pharyngeal muscles attached to the CPS and the body wall musculature of the prothorax. Several anastomoses exist between side branches of this nerve which were shown to contain motor pathways. The mesothoracic nerve (MeN) innervates a MH accessor and the longitudinal and transversal body wall muscles of the second segment.

See the light: electrophysiological characterization of the Bolwig organ's light response of Calliphora vicina 3rd instar larvae.

The anatomy and development of the larval cyclorraphous Diptera visual system is well established. It consists of the internal Bolwig organ (BO), and the associated nerve connecting it to the brain. The BO contributes to various larval behaviors but was never electrophysiologically characterized. We recorded extracellulary from the Bolwig nerve of 3rd instar Calliphora vicina larvae to quantify the sensory response caused by BO stimulation with light stimuli of different wavelengths, intensities and directions. Consistent with previous behavioral experiments we found the BO most sensitive to white and green, followed by blue, yellow, violet and red light. The BO showed a phasic-tonic response curve. Increasing light intensity produced a sigmoid response curve with an approximate threshold of 0.0105 nW/cm(2) and a dynamic range from 0.105 nW/cm(2) to 52.5 nW/cm(2). No differences exist between feeding and wandering larvae which display opposed phototaxis. This excludes reduced BO sensitivity from causing the switch in behavior. Correlating to the morphology of the BO frontal light evoked the maximal reaction, while lateral light reduced the neural response asymmetrically: Light applied ipsilaterally to the recorded BO always produced a stronger response than when applied from the contralateral side. This implies that phototacic behavior is based on a tropotactic mechanism.

The cephalic and pharyngeal sense organs of Calliphora vicina 3rd instar larvae are mechanosensitive but have no profound effect on ongoing feeding related motor patterns.

The anterior segments of cyclorraphous Diptera larvae bear various sense organs: the dorsal- and terminal organ located on the cephalic lobes, the ventral- and labial organs associated with the mouthplate and the internal labral organ which lies on the dorsal surface of the esophagus. The sense organs are connected to the brain via the antennal nerve (dorsal- and labral organ) or the maxillary nerve (terminal-, ventral-, labial organ). Although their ultrastructure suggests also a mechanosensory function only their response to olfactory and gustatory stimuli has been investigated electrophysiologically. Here we stimulated the individual organs with step-, ramp-, and sinusoidal stimuli of different amplitude while extracellulary recording their afferents from the respective nerves. The external organs show a threshold of approximately 2 microm. All organs responded phasically and did not habituate to repetitive stimuli. The low threshold of the external organs combined with their rhythmically exposure to the substrate suggested a putative role in the temporal coordination of feeding. We therefore repetitively stimulated individual organs while simultaneously monitoring the centrally generated motor pattern for food ingestion. Neither the dorsal-, terminal- or ventral organ afferents had an obvious effect on the ongoing motor rhythm. Various reasons explaining these results are discussed.

The brain can eat: establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster.

To establish the existence of a central pattern generator for feeding in the larval central nervous system of two Drosophila species, the gross anatomy of feeding related muscles and their innervation is described, the motor units of the muscles identified and rhythmic motor output recorded from the isolated CNS. The cibarial dilator muscles that mediate food ingestion are innervated by the frontal nerve. Their motor pathway projects from the brain through the antennal nerves, the frontal connectives and the frontal nerve junction. The mouth hook elevator and depressor system is innervated by side branches of the maxillary nerve. The motor units of the two muscle groups differ in amplitude: the elevator is always activated by a small unit, the depressor by a large one. The dorsal protractors span the cephalopharyngeal skeleton and the body wall hence mediating an extension of the CPS. These muscles are innervated by the prothoracic accessory nerve. Rhythmic motor output produced by the isolated central nervous system can simultaneously be recorded from all three nerves. The temporal pattern of the identified motor units resembles the sequence of muscle contractions deduced from natural feeding behavior and is therefore considered as fictive feeding. Phase diagrams show an almost identical fictive feeding pattern is in both species.

From behavior to fictive feeding: anatomy, innervation and activation pattern of pharyngeal muscles of Calliphora vicina 3rd instar larvae.

A description of the muscles and nerves involved in feeding of larval Calliphora vicina is given as a prerequisite to establish fictive feeding patterns recorded from the isolated central nervous system. Feeding Diptera larvae show a repetitive sequence of pro- and retraction of the cephalopharyngeal skeleton (CPS), elevation and depression of the mouth hooks and food ingestion. The corresponding pharyngeal muscles are protractors, mouth hook elevators and depressors, the labial retractor and cibarial dilator muscles. These muscles are innervated by the prothoracic accessory nerve (PaN), maxillary nerve (MN) and antennal nerve (AN) as shown electrophysiologically by recording action potentials from the respective nerve that correlate to post-synaptic potentials on the muscles. All three nerves show considerably more complex branching patterns than indicated in the literature. Extracellular recordings from the stumps of PaN, MN and AN connected to an isolated CNS show spontaneous rhythmic motor patterns that reflect the feeding sequence in intact larvae. Variability of the feeding pattern observed in behavioral experiments is also evident from the level of motor output from an isolated CNS. The data obtained from Calliphora will facilitate electrophysiological investigations dealing with the genetic background of feeding behavior in Drosophila larvae.

Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicina third instar larvae.

The stomatogastric nervous system (SNS) associated with the foregut was studied in 3rd instar larvae of Drosophila melanogaster and Calliphora vicina (blowfly). In both species, the foregut comprises pharynx, esophagus, and proventriculus. Only in Calliphora does the esophagus form a crop. The position of nerves and neurons was investigated with neuronal tracers in both species and GFP expression in Drosophila. The SNS is nearly identical in both species. Neurons are located in the proventricular and the hypocerebral ganglion (HCG), which are connected to each other by the proventricular nerve. Motor neurons for pharyngeal muscles are located in the brain not, as in other insect groups, in the frontal ganglion. The position of the frontal ganglion is taken by a nerve junction devoid of neurons. The junction is composed of four nerves: the frontal connectives that fuse with the antennal nerves (ANs), the frontal nerve innervating the cibarial dilator muscles and the recurrent nerve that innervates the esophagus and projects to the HCG. Differences in the SNS are restricted to a crop nerve only present in Calliphora and an esophageal ganglion that only exists in Drosophila. The ganglia of the dorsal organs give rise to the ANs, which project to the brain. The extensive conformity of the SNS of both species suggests functional parallels. Future electrophysiological studies of the motor circuits in the SNS of Drosophila will profit from parallel studies of the homologous but more accessible structures in Calliphora.

Anatomical and functional characterisation of the stomatogastric nervous system of blowfly (Calliphora vicina) larvae.

The anatomy and functionality of the stomatogastric nervous system (SNS) of third-instar larvae of Calliphora vicina was characterised. As in other insects, the Calliphora SNS consists of several peripheral ganglia involved in foregut movement regulation. The frontal ganglion gives rise to the frontal nerve and is connected to the brain via the frontal connectives and antennal nerves (ANs). The recurrent nerve connects the frontal- to the hypocerebral ganglion from which the proventricular nerve runs to the proventricular ganglion. Foregut movements include rhythmic contractions of the cibarial dilator muscles (CDM), wavelike movements of crop and oesophagus and contractions of the proventriculus. Transections of SNS nerves indicate mostly myogenic crop and oesophagus movements and suggest modulatory function of the associated nerves. Neural activity in the ANs, correlating with postsynaptic potentials on the CDM, demonstrates a motor pathway from the brain to CDM. Crop volume is monitored by putative stretch receptors. The respective sensory pathway includes the recurrent nerve and the proventricular nerve. The dorsal organs (DOs) are directly connected to the SNS. Mechanical stimulation of the DOs evokes sensory activity in the AN. This suggests the DOs can provide sensory input for temporal coordination of feeding behaviour.

Juvenile hormone-dependent motor activation in the adult locust Locusta migratoria.

Abdominal motoneurones of the locust Locusta migratoria were investigated in immature, mature and allatectomised females to compare their response characteristics during reproductive development. These motoneurones were chosen because they control muscles which are involved in extreme lengthening during egg-laying behaviour. The study focused on changes in motoneurone firing activity and its possible regulation by juvenile hormone. In isolated nerve-muscle preparations, increased resting motor activity was found in mature (>14 days) but not in immature females (<5 days). Removing the corpora allata, the gland producing juvenile hormone in insects, prevented increased motor activity. Stimulus evoked activation of the motor system led to a characteristic burst of action potentials which lasted for a few seconds. The time-course and amount of activation changed significantly during reproductive development. Mature females displayed longer lasting and higher activity than immature or allatectomised females, but only those segments involved in egg-laying were found to express the altered firing properties. Single cell analysis of motoneurone dendritic morphology or membrane properties revealed no evidence that could be causative for the activity changes seen during reproductive development. The results suggest that altered motoneurone activity serves to adapt females to the neuromuscular requirements of egg-laying behaviour.