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Journal of Insect Physiology 53 (2007) 349–360 Anatomical and functional characterisation of the stomatogastric nervous system of blowfly (Calliphora vicina) larvae Andreas Schoofs, Roland SpieX Institut fu ¨ r Zoologie, Abteilung Neurobiologie, Universita ¨ t Bonn, Poppelsdorfer SchloX , 53115 Bonn, Germany Received 9 November 2006; received in revised form 20 December 2006; accepted 21 December 2006 Abstract 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. r 2007 Elsevier Ltd. All rights reserved. Keywords: Blowfly larva; Foregut; Insect nervous system; Afferent–efferent pathway 1. Introduction For over two centuries the stomatogastric nervous system (SNS) of crustaceans is established as a model system for rhythmic motor pattern generation and for the underlying neuronal circuits (Harris-Warwick et al., 1992; Selverston and Moulins, 1987). Compared to this model system, little is known about the functionality and neuronal pathways in the insect SNS, especially the SNS of larval dipteran species. Anatomical data on the insect SNS are available for several insect groups such as adult Caelifera (Aubele and Klemm, 1977; Ayali et al., 2002; Burrows, 1996), adult Blattodea (Gundel and Penzlin, 1978, 1980; Willey, 1961), larval Lepidoptera (Bell et al., 1974; Miles and Booker, 1994; Sasaki and Asaoka, 2006) and larval Hymenoptera (Boleli et al., 1998). The anatomical data available for Diptera (Drosophila) are based on developmental investi- gations on late embryos and early first instar larvae (Forjanic et al., 1997; Gonzalez-Gaitan and Ja¨ckle, 1995, 2001; Hartenstein, 1997; Hartenstein et al., 1994). Anato- mical data on the larval Calliphora SNS are fragmentary and restricted to the retro-cerebral complex (Cantera, 1988; Cantera et al., 1994). The insect SNS is composed of the following elements: The frontal ganglion (FG), located on the dorsal surface of the oesophagus, is connected to the ARTICLE IN PRESS www.elsevier.com/locate/jinsphys 0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.12.009 Abbreviations: AMN, accessory muscle nerve; AN, antennal nerve; BH, brain hemispheres; CDM, cibarial dilator muscle; CN, crop nerve; CPS, cephalo-pharyngeal skeleton; DA, dorsal arm; DO, dorsal organ; DOG, dorsal organ ganglion; FC, frontal connective; FG, frontal ganglion; FN, frontal nerve; HCG, hypocerebral ganglion; IOE, innervation oesophagus; MH, mouth hooks; NCS, nervus cardiostomatogastricus; OE, oesopha- gus; PH, pharynx; PV, proventriculus; PVG, proventricular ganglion; PVN, proventricular nerve; RG, ring gland; RN, recurrent nerve; SNS, stomatogastric nervous system; SOG, suboesophagial ganglion; SR, sphincter region; TC, tritocerebrum; TO, terminal organ; TOG, terminal organ ganglion Corresponding author. Tel.: +49 228 735495. E-mail address: [email protected] (R. SpieX).

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

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Page 1: Anatomical and functional characterisation of the stomatogastric nervous system of blowfly (Calliphora vicina) larvae

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Journal of Insect Physiology 53 (2007) 349–360

0022-1910/$ - se

doi:10.1016/j.jin

Abbreviations

brain hemisphe

cephalo-pharyn

dorsal organ ga

frontal nerve; H

MH, mouth ho

gus; PH, phary

PVN, proventri

stomatogastric

sphincter region

organ ganglion�CorrespondE-mail addr

www.elsevier.com/locate/jinsphys

Anatomical and functional characterisation of the stomatogastricnervous system of blowfly (Calliphora vicina) larvae

Andreas Schoofs, Roland SpieX�

Institut fur Zoologie, Abteilung Neurobiologie, Universitat Bonn, Poppelsdorfer SchloX , 53115 Bonn, Germany

Received 9 November 2006; received in revised form 20 December 2006; accepted 21 December 2006

Abstract

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.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Blowfly larva; Foregut; Insect nervous system; Afferent–efferent pathway

1. Introduction

For over two centuries the stomatogastric nervoussystem (SNS) of crustaceans is established as a modelsystem for rhythmic motor pattern generation and for theunderlying neuronal circuits (Harris-Warwick et al., 1992;Selverston and Moulins, 1987). Compared to this model

e front matter r 2007 Elsevier Ltd. All rights reserved.

sphys.2006.12.009

: AMN, accessory muscle nerve; AN, antennal nerve; BH,

res; CDM, cibarial dilator muscle; CN, crop nerve; CPS,

geal skeleton; DA, dorsal arm; DO, dorsal organ; DOG,

nglion; FC, frontal connective; FG, frontal ganglion; FN,

CG, hypocerebral ganglion; IOE, innervation oesophagus;

oks; NCS, nervus cardiostomatogastricus; OE, oesopha-

nx; PV, proventriculus; PVG, proventricular ganglion;

cular nerve; RG, ring gland; RN, recurrent nerve; SNS,

nervous system; SOG, suboesophagial ganglion; SR,

; TC, tritocerebrum; TO, terminal organ; TOG, terminal

ing author. Tel.: +49228 735495.

ess: [email protected] (R. SpieX).

system, little is known about the functionality andneuronal pathways in the insect SNS, especially the SNSof larval dipteran species.Anatomical data on the insect SNS are available for

several insect groups such as adult Caelifera (Aubele andKlemm, 1977; Ayali et al., 2002; Burrows, 1996), adultBlattodea (Gundel and Penzlin, 1978, 1980; Willey, 1961),larval Lepidoptera (Bell et al., 1974; Miles and Booker,1994; Sasaki and Asaoka, 2006) and larval Hymenoptera(Boleli et al., 1998). The anatomical data available forDiptera (Drosophila) are based on developmental investi-gations on late embryos and early first instar larvae(Forjanic et al., 1997; Gonzalez-Gaitan and Jackle, 1995,2001; Hartenstein, 1997; Hartenstein et al., 1994). Anato-mical data on the larval Calliphora SNS are fragmentaryand restricted to the retro-cerebral complex (Cantera, 1988;Cantera et al., 1994). The insect SNS is composed of thefollowing elements: The frontal ganglion (FG), located onthe dorsal surface of the oesophagus, is connected to the

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central nervous system via the paired frontal connectives(FCs). The unpaired recurrent nerve projects from the FGto the hypocerebral ganglion (HCG), which is connected tothe proventricular ganglion (PVG) by the proventricularnerve. The function of the SNS is generally associated withthe generation of foregut motor patterns.

The insect foregut consists of the pharynx with thecibarial pump for food intake and the oesophagus, whichtransports the food to the midgut. In some species a cropexists, which serves as temporary reservoir for ingestedmaterial. In larval Lepidoptera dilator muscles, attached tothe roof of the oesophagus, mediate food ingestion (Milesand Booker, 1998; Sasaki and Asaoka, 2006). The motorpatterns of the oesophagus, participating in food transport,include peristaltic waves and synchronous contractions(Duve et al., 1999; Gelperin, 1966; Miles and Booker,1994). Food passage from foregut to midgut is regulated bythe muscular proventriculus (Davey and Treherne, 1963b;Zinke et al., 1999).

The insect SNS is involved in the generation of foregutmotor activity associated with feeding or air swallowingduring the moult (Bestman and Booker, 2003; Carlsonand Gara, 1983; Griss et al., 1991; Hill et al., 1966; Milesand Booker, 1994, 1998; Zilberstein and Ayali, 2002;Zilberstein et al., 2006). Recent studies demonstratethat the FG contains the motor neurons and neural circuitsof the pattern generator required for rhythmic foregutmovements in adult locusts (Ayali, 2004; Ayali andZilberstein, 2004; Ayali et al., 2002; Zilberstein and Ayali,2002) and Manduca sexta larvae (Miles and Booker, 1994,1998). Sensory components of the SNS are described foradult blowflies where stretch receptors in the foregutcontribute to the regulation of food intake (Gelperin,1967, 1971a, b).

Virtually nothing is known about efferent and afferentpathways in the SNS of larval Diptera. We have thuschosen to investigate the large third-instar larvae ofCalliphora vicina. This species makes a promisingmodel organism to study the functionality of the SNS ofdipteran larvae because the foregut structures withthe associated SNS are easily accessible for electrophysio-logical experiments. The relatively close phylogeneticrelation to Drosophila suggests that data obtained fromCalliphora on SNS functionality will facilitate comparableinvestigations in the considerably smaller Drosophila

larvae. Future studies on Drosophila larvae might thereforebe able to combine electrophysiological and genetictechniques.

In the present study, we investigated the gross anatomyof the SNS associated with the foregut in third-instar larvaeof C. vicina. The basic foregut movement patterns for foodingestion and transport are described. To gain first insightinto SNS functionality the effect of nerve transections onforegut movements were studied. Efferent (motor) path-ways that regulate cibarial dilator musculature (CDM)contractions and afferent (sensory) pathways from the cropand the dorsal organs (DOs) are established.

2. Material and methods

2.1. Animals

Only late third-instar larvae of C. vicina from our owncolony were used for the experiments. Blowfly larvaeobtained from a local fishing supply were allowed topupate. The hatched flies were selected for C. vicina, kept instandard cages (16� 9� 13 cm, 25 flies per cage) andsupplied with sugar water, oatmeal and milk powder. Theydeposited their eggs in 50ml beakers filled with groundmeat. The hatched larvae were transferred to fresh meatevery three days until they reached the third-larval stage.The animals used for experiments were cleaned and kept at4 1C on paper tissue until preparation.

2.2. Preparation

The larvae were dissected in a Petri dish lined with clearsilicone (Wacker Silicones, Elastosil RT 601). The wholepreparation was covered with Calliphora saline [(in mM:NaCl: 172; KCl: 2.5; CaCl2: 0.5, MgCl2: 8; NaHCO3: 0.6;NaH2CO3: 0.3; PH: 7.2 (Magazanik and Fedorova, 2003)].The larvae were pinned down with fine insect needles andopened along the dorsal midline. Care was taken not todamage the cephalo-pharyngeal skeleton (CPS). The cuticlewas pinned down laterally. The fat body and the salivaryglands were removed. In this stage of preparation allcomponents of the SNS except the FG with the frontalnerve (FN) were visible. To visualise the projections of finenerve branches they were stained with brilliant blue (Sigma1% in Calliphora saline). The solution was topicallyapplied to the region of interest for 30 s and thepreparation was subsequently washed with fresh saline.The anatomical features were photographed or drawn byhand and reconstructed for the overall view presented inFig. 1.

2.3. Foregut movements

In the intact larva the CDM was enclosed by the CPS,which made it difficult to measure the contraction force ofthe muscles. We thus chose to measure the shift ofmembrane potential during contractions by intracellularmuscle recordings (details see below). For this the CPS wasdissected free and the connective tissue between the dorsalarms (DAs) was cut without damaging the neural supply ofthe muscles. The DAs were pinned down laterally whichexposed the muscle bundles of the CDM and made themeasily accessible to intracellular recordings.The movement pattern of the crop and the proventricu-

lus were studied in preparations with fully intact SNS.Larvae were pinned down dorsal side up, opened along themidline and only the fat body and salivary glands wereremoved. The crop and the proventriculus were stainedwith brilliant blue. To enhance contrast, a small piece ofwhite plastic foil was placed under the crop and the

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Fig. 1. Anatomy of the foregut and associated stomatogastric nervous system. (A) Semi-schematic drawing of the foregut structures with the associated

SNS. Abbreviations: AMN, accessory muscle nerve; AN, antennal nerve; BH, brain hemispheres; CDM, cibarial dilator muscle; CN, crop nerve; CPS,

cephalo-pharyngeal skeleton; DA, dorsal arm; DO, dorsal organ; DOG, dorsal organ ganglion; FC, frontal connective; FG, frontal ganglion; FN, frontal

nerve; HCG, hypocerebral ganglion; IOE, innervation oesophagus; MH, mouth hooks; NCS, nervus cardio-stomatogastricus; OE, oesophagus; PH,

Pharynx; PV, proventriculus; PVG, proventricular ganglion; PVN, proventricular nerve; RG, ring gland; RN, recurrent nerve; SOG, suboesophagial

ganglion; SR, sphincter region; TC, tritocerebrum; TO, terminal organ; TOG, terminal organ ganglion. (B) Schematic drawing of the motor/efferent

(green) and sensory/afferent (black) pathways in the Calliphora SNS. Small arrows indicate the direction of action potentials. The NCS and the ring gland

are not shown.

A. Schoofs, R. SpieX / Journal of Insect Physiology 53 (2007) 349–360 351

proventriculus. The movements were then filmed with adigital camera (Fuji Finepix S9500) mounted to thedissecting microscope. Films were stored on computer,cut and the sequences of representative movements weretransformed to single frames (frame to frame: 0.8 s). Theimages of the frames were digitally adapted (Image J). The

foregut movements of 20 larvae were filmed and analysed.The relative amplitude of the proventriculus contractionwas calculated with Image J: the diameter of the anteriorpart of the proventriculus before the contraction wasdefined as 100%. The movement patterns of the foregut areshown in Fig. 2.

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Fig. 2. Movement patterns of the foregut. (A) Left: experimental set-up,

with a lateral view of the cephalo-pharyngeal skeleton and the position of

the intracellular electrodes. Right: Potentials recorded from an anterior

(L2) and a posterior (R6) muscle bundles show the successional activity of

cibarial dilator muscle bundles. (B) Sequence of a peristaltic contraction

(arrows) that propagates from the distal part of the crop to the

oesophagus (OE). The crop contains an air bubble (AB). (C) Sequence

of the contraction of the anterior proventriculus musculature followed by

a telescopic ‘‘lift’’.

A. Schoofs, R. SpieX / Journal of Insect Physiology 53 (2007) 349–360352

2.4. Lesion experiments

In these experiments the impact of nerve transection onthe movement frequency of the oesophagus, crop andproventriculus was studied. The larvae were opened alongthe dorsal midline and the crop and proventriculus weredissected free. The preparation was given 5min to express abaseline activity. After that the movements of the crop andoesophagus (peristaltic waves and contractions) and theproventriculus (contractions) were observed and countedfor 3min. Then either the PVG was severed from theproventriculus (lesion 1) or the proventricular nerve wascut posterior to the HCG (lesion 2). Two minutes after therespective transection the movements of the crop, oeso-phagus and proventriculus were again counted for three

minutes. In one animal either lesion 1 or lesion 2 wasapplied. During the whole experiment the preparation wassuperfused with fresh oxygenated saline. A total of 25larvae were used for each lesion experiment. The resultingdata of each movement category were summarised andtested for significant differences with a paired student t-test(significance level as indicated in Fig. 3: **pp0.005,***pp0.0005).

2.5. Intracellular recordings from the cibarial dilator

musculature

The muscle bundles of the CDM were exposed asdescribed above. Glass electrodes (filled with 3M KCl, tipresistance 20–30MO pulled from thinwalled borosilicateglass, TW 100F-4, World Precision Instruments USA) wereused to impale the muscle bundle of choice near itsattachment site to the DAs. The electrodes were designedto have an extraordinary long shaft, which ensured thenecessary flexibility to follow small muscle contractions.With this configuration, stable recordings were possible forover 30min. The electrode was connected to a custom-made intracellular amplifier (Elektronikwerkstatt Bota-nisches Institut, University of Bonn, Germany, 100�amplification).

2.6. Electrical stimulation

The antennal nerve (AN) was dissected free under salineand a small piece of Parafilm was placed underneath it.A small petroleum jelly pool was built around thenerve using a syringe pulled to a fine tip. The saline levelwas then lowered below the rim of the pool, thus isolatingthe lumen of the pool from the surrounding saline.A 200 mm silver wire, connected to a stimulus isolator(world precision instruments, A360D-C), was placed in thelumen of the pool. Square pulses (3V, 1ms) triggeredwith a stimulus generator (CED 1401 micro) were appliedto the nerve.

2.7. Extracellular recordings

Extracellular recordings were obtained from the anten-nal-, the recurrent-, and the proventricular nerve. Thenerve of choice was dissected free and a petroleum jellypool was built around it. A 200 mm silver wire (differentelectrode) connected to a custom made preamplifier(Michael Hofmann) was then placed in the pool. Theindifferent electrode was placed in the surrounding saline.To obtain information on conduction direction of theaction potentials and neural pathways, double recordingsfrom the same or different nerves were obtained simulta-neously. The recorded action potentials were filtered (highpass: 3 kHz, low pass 100Hz), recorded and stored in realtime using a 4-channel A-D board (CED 1401 micro) andprocessed with Spike2.

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Fig. 3. Influence of SNS lesions on movement frequency of different foregut structures: (A–C) Lesion one (L1) significantly affects the contraction rates of

proventriculus, crop and oesophagus. (D) Lesion two (L2) also causes a significant frequency reduction of oesophagus movements. None of the described

movements ceases after the lesions.

A. Schoofs, R. SpieX / Journal of Insect Physiology 53 (2007) 349–360 353

2.8. Manipulation of crop volume

To increase the crop volume by injecting saline, a smallhole was cut into the most distal part of the crop. Thetip of a fine tube, pulled from a 1ml syringe, was insertedinto the hole. The crop was tied to the tube with a fibreof dental floss. The plunger of the syringe was movedwith a micrometre screw, allowing a fine adjustmentof the crop volume. The micrometre screw was connectedto a potentiometer, which produced voltage changesproportional to the volume changes of the crop. Noquantitative experiments were performed. To demonstratesensory pathways, the crop was just considered to beempty or full.

2.9. Stimulation of the dorsal organ

To demonstrate the mechanosensory function of theDOs, extracellular recordings were obtained from the ANjust anterior to the brain while the DO was mechanicallystimulated. A glass electrode with broken tip mounted on amicromanipulator was used to dislocate the most distalpart (dome) of the DO. The movements of the manipulatorwere monitored with a potentiometer. Action potentialsevoked by mechanical stimulation were recorded and

stored in real time using a 4-channel A-D board (CED1401 micro) and processed with Spike2.

3. Results

3.1. Anatomy of the foregut

A semi-schematic drawing of the foregut structures andthe associated SNS of third-instar Calliphora larvae is givenin Fig. 1. The larval foregut consisted of the pharynx (PH)with the CPS, the oesophagus (OE, diameter ca. 180 mm),the expandable crop and the proventriculus (PV, diameterca. 600 mm). The CPS (length ca. 1250 mm) served asattachment site for complex musculature (not shown inFig. 1) moving the mouth hooks (MHs) and the CPSrelative to the body during feeding. The cibarial part of theoesophagus was enclosed by the sclerites of the CPS. TheCDM spanned the DAs of the CPS and the roof of thecibarial part of the oesophagus. The crop protruded fromthe oesophagus between the CPS and the brain. Its distalpart formed an expandable contractile pouch, where freshfood was stored before passed on in small portions. Thefood passage from the crop into the oesophagus wascontrolled by the sphincter region (SR). Posterior to thecrop, the oesophagus passed through a gap between the

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brain hemispheres (BH) until it reached the proventriculus,which controlled the food passage into the midgut.

3.2. Anatomy of the stomatogastric nervous system

associated with the foregut

The FG (ca. 60� 60 mm) was located anterior to thebrain on the dorsal surface of the cibarial part of theoesophagus. Its shape resembled a rhombus with a nerveemerging from each corner: the recurrent nerve (RN,diameter ca. 25 mm); the FCs (diameter ca. 25 mm) and theFN (diameter ca. 25 mm). The FN projected anteriorly toinnervate the CDM. From each FC, a small nerve(AMN—accessory muscle nerve) arose to innervate CPSassociated muscles. Further distal, the FCs fused with theAN. The recurrent nerve ran along the oesophagus until itreached the HCG (ca. 150� 60 mm). Posterior to the CPSthe crop nerve (CN) split from the recurrent nerve andprojected along the surface of the crop. The oesophaguswas innervated in the same region (IOE—innervationoesophagus) by several small nerves that originated fromthe recurrent- and CN. The diffuse HCG, located dorsallyto the suboesophagial ganglion (SOG), was connected tothe ring gland (RG) by the nervus cardiostomatogastricus(NCS). The roughly triangular shaped PVG (ca.160� 70 mm) was attached to the anterior rim of theproventriculus (PV) and was connected to the HCG by theproventricular nerve (PVN, diameter ca. 25 mm).

The DOs were not a part of the foregut, but showeddirect neuronal connections to the SNS. They were locatedon the cephalic lobe of the larvae and served sensoryfunctions. They consisted of a dome-like structure in acylindrical socket (diameter ca. 45 mm), embedded in thecuticle of the cephalic lobe. The ganglia of the dorsalorgans (DOG) were connected to the tritocerebrum via theANs.

3.3. Movement patterns of the foregut

The foregut served to ingest food (pharynx), store it(crop), transport it (oesophagus) and control food passageinto the midgut (proventriculus). It could thus be expectedthat these foregut regions work co-ordinately to optimisefood flow. Yet, as consistently observed, the activity of thenamed foregut structures did not show any obvioustemporal correlation or synchronisation.

The cibarial dilator musculature was organised in pairsof laterally symmetrical muscle bundles. During periods ofrhythmically activity their contractions (interval ca. 0.7 s)increased the volume of the cibarial part of the oesophagusto ingest food. The muscle bundles did not contractsimultaneously: the anterior muscles showed an earlierand prolonged activation compared to the posterior ones(Fig. 2). This motor pattern caused a peristaltic expansionof the oesophagus within the CPS.

In freshly fed larvae the crop was visible through thebody wall and it could reach up to one third of the body

length. The pouch filled with food and the adjacentsphincter region showed complex contraction patterns.Food was dispersed in the crop by wavelike movementsthat arose in the distal part of the pouch and travelledtoward the sphincter region and vice versa. Small foodportions were occasionally propelled into the oesophagusby a peristaltic wave that propagated across the sphincterregion onto the oesophagus (Fig. 2). Normally, a contrac-tion of the oesophagus anterior to the sphincter regionensured that food was only passed posteriorly. Peristalticwaves on the oesophagus were observed travelling poster-iorly toward the proventriculus and anteriorly toward thecrop. Additionally, sections of variable length (up to theentire oesophagus) could either be compressed by contrac-tion of the ring musculature or be jolted by contractions oflongitudinal muscles.Two basic movement patterns of the muscular PV were

involved in the regulation of food passage from theoesophagus to midgut (Fig. 2). First, contractions of thering musculature in the anterior part of the PV couldconstrict the oesophagus. The magnitude of these PVcontractions ranged from weak and hardly visible, tostrong contractions that reduced the diameter of theanterior PV region by up to 60% and extended over theanterior half of the PV. Second, a contraction of long-itudinal muscles of the PV resulted in an anterior directedmovement of the PV, along the oesophagus. We refer tothis type of movement as ‘‘lift phase’’ of the PV.Occasionally the ring musculature constricted the oeso-phagus at the highest point of the lift phase.

3.4. Lesion experiments

To gain first insight into the functionality of the SNS,lesions were applied at two different locations. Severing thePVG from the proventriculus (lesion 1, Fig. 3) significantlyreduced the baseline activity of crop, oesophagus andproventriculus. Crop activity was reduced by 36% from2.370.4 to 1.270.1 per min (Student’s t-test: p ¼ 0.003,n ¼ 25). Movements of the oesophagus were reduced by79% from 2.170.9 to 0.4570.02 per min (Student’s t-test:p ¼ 0.0003, n ¼ 25). The frequency of proventriculuscontractions was reduced by 38% from 2.170.1 to1.370.1 per min (Student’s t-test: p ¼ 0.0003, n ¼ 25).Cutting the proventricular nerve between hypocerebral-

and PVG (lesion 2, Fig. 3) reduced the frequency ofoesophagus movements by 50% from 2.870.7 to 1.470.1per min (Student’s t-test: p ¼ 0.0002, n ¼ 25). This lesionhad no significant effect on crop and proventriculusactivity.None of the movements investigated completely stopped

after nerve transection.

3.5. Efferent (motor) pathways

Cutting the ANs anterior to the brain immediatelystopped the visible contractions of the cibarial dilator

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ARTICLE IN PRESSA. Schoofs, R. SpieX / Journal of Insect Physiology 53 (2007) 349–360 355

musculature while electrical stimulation of the AN evokedvisible twitches of the CDM. Intracellular recordings fromthe muscle fibres revealed postsynaptic potentials (PSPs)that showed a temporal correlation with the electricalstimulus. They had a duration of approximately 70ms andranged in amplitude from 2 to 5mV (Fig. 4). The delayfrom onset of the stimulus to the PSP was 5ms. Theamplitude of the PSP to a given stimulus could differ.These results qualitatively demonstrated that axons ofmotor neurons in the AN project onto the CDM via theFCs, the FG and the FN.

Fig. 4. Motor pathway: activation of cibarial dilator musculature. (A) Experim

frontal ganglion while recording from the CDM. (B) Postsynaptic potentials

potentials are superimposed. *: Stimulus artefact.

Fig. 5. Motor activity in the antennal nerve. (A) Experimental set-up: two int

musculature (RCDM, LCDM) and two extracellular recordings from both ant

were consecutively cut between the brain and the recording site (L1, L2). (B) Th

the CDM contractions. RCDM, LCDM: intracellular recordings from the CDM

antennal nerve innervates IPSI- and contralateral CDM. After the first lesi

contractions of both CDM proceed. The shaded areas indicate the electrical i

Simultaneous intracellular recordings from the CDM(Fig. 5) and extracellular recordings from the AN showedthat the PSPs recorded from the CDM correlated withbursts of action potentials in the AN. These burstsoccurred synchronous in both ANs, as did the PSPs incontralateral muscle bundles. Motor axons in one ANactivated both, lateral and contralateral CDM bundles.This was demonstrated with simultaneous recordings fromboth ANs (RAN, LAN) and contralateral CDM bundles(RCDM, LCDM), followed by successive transection ofthe ANs. Cutting the right AN anterior to the brain

ental set-up: electrical stimulation of the antennal nerve between brain and

on the CDM evoked by electrical stimulation of the antennal nerve. Five

racellular recordings from opposing muscle bundles of the cibarial dilator

ennal nerves (RAN, LAN) were obtained simultaneously. Antennal nerves

e motor activity in both antennal nerves is synchronous and correlates with

. RAN, LAN: extracellular recordings form the antennal nerves. (C) One

on (L1) the bursts in RAN cease, but motor activity in LAN and the

nterferences caused by cutting the nerves.

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immediately abolished the bursts of action potentials inthis nerve. The bursts in the remaining contralateral ANand both CDM bundles proceeded uninfluenced. Cuttingthe second AN abolished all postsynaptic potentials in theCDM. The fact that the motor bursts ceased after cuttingthe AN anterior to the brain strongly suggested that thecells bodies of the corresponding motor neurons werelocated in the brain.

3.6. Afferent (sensory) pathways

Manipulation of the crop volume demonstrated theexistence of sensory structures, probably stretch receptorsembedded in the muscle tissue of the crop (Fig. 6).Stretching the crop by saline infusion increased neuralactivity in the recurrent- and proventricular nerve.A higher temporal resolution of the recording revealedthat identical action potentials occurred in both nerveswith a delay of 9ms. The sensory pathway therefore ran

Fig. 6. Sensory pathway: crop volume. (A) Experimental set-up: manipulation

were performed, the crop was just considered to be empty (E) or full (F). (B) Sim

sensory activity apparent in the recurrent nerve also occurs in the proventricul

nerve. The neural activity in the antennal nerve does not depend on crop volu

from the CN over the recurrent nerve through the HCGinto the proventricular nerve.A possible pathway for sensory information about crop

volume to the central nervous system was via the recurrentnerve, the FCs and the ANs. Surprisingly, while a full cropelicited a sensory response in the recurrent nerve noresponse was present in the AN.The DOs were directly connected to the SNS and

therefore provided a potential source for sensory inputto the SNS. During feeding and crawling, the larvarhythmically extended the CPS and the cephalic lobe,exposing the DOs to the surrounding substrate (Fig. 7).Our results demonstrate that dislocating the dome of theDO by 10 mm elicit a burst of action potentials in the AN.Pushing the dome into the socket increased the actionpotential frequency as did releasing the dome. Thisindicated that the DOs might also contribute mechan-osensory information to the SNS monitoring feedingmovements.

of crop volume by injection of ringer solution. No quantitative experiments

ultaneous recording from the recurrent and the proventricular nerve. The

ar nerve. (C) Simultaneous recording from the recurrent and the antennal

me. The black bars indicate the section shown in higher time resolution.

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ARTICLE IN PRESS

Fig. 7. Sensory pathway: dorsal organ serves as mechano-receptor. (A)

Drawing of the anterior aspect of an intact larva with extended cephalic

lobe. Note the exposed dorsal organ (arrow). (B) Experimental set-up: the

dome of the dorsal organ was dislocated by 10mm. The dimensions of the

drawn dorsal organ are in scale. Recordings were obtained from the

ipsilateral antennal nerve. The antennal nerve was cut anterior to the brain

to abolish motor activity. (C) AN: recording from the antennal nerve

showing the sensory response to mechanical stimulation of the dorsal

organ. Both, pushing the dome into the socket (P) and releasing it (R)

results in increased neural activity.

A. Schoofs, R. SpieX / Journal of Insect Physiology 53 (2007) 349–360 357

4. Discussion

4.1. Anatomy of foregut

In Calliphora larvae the pharynx consists of the CPS andthe associated musculature. Food ingestion is mediated byrhythmic contractions of the CDM. During metamorpho-sis the simple pharyngeal organisation is lost: in adult fliesan extendable proboscis with complex musculature exists(Mier et al., 1985; Rajashekhar and Singh, 1994; Starre andRuigrok, 1980). In Drosophila larvae no crop exists, but theoesophagus runs as a straight tube from the pharynx to theproventriculus (Hartenstein, 1997; Zinke et al., 1999),which controls the food passage from the oesophagus intothe midgut (Davey and Treherne, 1963b; Zinke et al.,1999). The morphology of the larval proventriculus ofCalliphora is similar to that described for Drosophila

(Aggarwal and King, 1967).

4.2. Anatomy of the SNS associated with the foregut

The FG of Calliphora is located on the dorsal surface ofthe oesophagus, which is in accordance with the anatomi-cal data available for other insect orders (Ayali, 2004;Boleli et al., 1998; Gundel and Penzlin, 1978; Kirby et al.,1984; Miles and Booker, 1994, 1998). The FG of lateembryos/early larvae of Drosophila was described as ‘‘tworather asymmetrical half-ganglia’’ (Campos-Ortega andHartenstein, 1985; Hartenstein, 1997). So possibly, theshape of the FG differs between early Drosophila and lateCalliphora larvae or structural modifications occur during

ongoing larval development. Inconsistencies appear in thenomenclature of the FCs: in other insect orders this termwas used for the nerves that connect the FG to the brain. InCalliphora larvae the corresponding nerve fuses with thenerve that connects the DOs to the brain. In Drosophila

larvae that nerve is labelled as AN (Cobb, 1999; Pythonand Stocker, 2002a; Tissot et al., 1997). To assureconformity, we adopted the nomenclature in use forDrosophila. According to this nomenclature the frontalconnectives connect the antennal nerves to the frontalganglion.In larval Calliphora the recurrent nerve innervates the

oesophagus and gives rise to the crop nerve. A correspond-ing crop innervation exists in adult blowflies (Richer et al.,2000). Our data on the HCG are in accordance with theanatomical situation described for larval Calliphora

(Cantera, 1988; Cantera et al., 1994) and late Drosophila

embryos (Campos-Ortega and Hartenstein, 1985; Forjanicet al., 1997; Gonzalez-Gaitan and Jackle, 1995; Hartensteinet al., 1994). As in larval Drosophila, the proventricularnerve connects the HCG to the PVG, which is attached tothe anterior rim of the proventriculus (Campos-Ortega andHartenstein, 1985; Forjanic et al., 1997; Gonzalez-Gaitanand Jackle, 1995; Hartenstein, 1997; Hartenstein et al.,1994).

4.3. Movement patterns of the foregut

Blowfly larvae live and feed on decaying meat andtherefore ingest a rather liquid food. Thus, the CDM serveas ‘‘suction pump’’ comparable to the cibarial pumpdescribed for other liquid feeding insects (Miles andBooker, 1998; Sasaki and Asaoka, 2006). A contractionof the CDM increases the volume of the cibarial section ofthe oesophagus thus mediating food ingestion. Theperistaltic expansion of the oesophagus caused by earlierand prolonged activity of the more anterior CDM may bean adaptation for optimising food intake. As in adultblowflies (Gelperin, 1966), food ingested by CDM activitywas stored in the crop, and later, small portions werepropelled through the oesophagus towards the midgut. Theperistaltic waves that travelled in both directions over thecrop and the oesophagus show striking similarity withforegut movements described for other insects (Cook et al.,1969; Duve et al., 1999; Kirby et al., 1983; Miles andBooker, 1994; Yeager, 1931).It can be assumed that the contractions of the anterior

part of the proventriculus constrict the oesophagus to limitthe amount of food transported into the midgut. The ‘‘lift’’of the proventriculus, together with a contraction of theanterior part resembles swallowing movements, whichensure a regulated food flow into the midgut.

4.4. Lesions experiments

As mentioned in the introduction, virtually nothing isknown about how and where foregut movements are

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generated in dipterous larvae. The anatomy of the foregutand the associated nervous system suggests that thedescribed movements of the crop, oesophagus andproventriculus are under motor control of the centralnervous system and/or the SNS. Given that such motorpathways exist, it could be expected that first, contractionsof the oesophagus, crop and proventriculus correlate withneural activity in the antennal-, recurrent- or proventri-cular nerve. This was never confirmed by extracellularrecordings from the respective nerves. Second, the appliedlesions should only affect the contraction rate of theproventriculus because they leave the anterior componentsof the CNS and SNS intact. Yet, disconnecting the PVGfrom the proventriculus (lesion 1) significantly reduces thebaseline activity of the oesophagus, the crop and theproventriculus, and cutting the proventricular nerve (lesion2) reduces the contraction rate of the oesophagus, butneither lesion completely abolishes the observed move-ments. Lesion 1 demonstrates that unlike in adultcockroaches (Davey and Treherne, 1963a; Engelmann,1968), the PVG is not necessary for the generation ofproventriculus contractions, suggesting a myogenic activa-tion of the proventriculus musculature. A comparablesituation is described for the foregut of crickets (Mohl,1972), cockroaches (Cook et al., 1969) and the oviductmuscles in locusta, (Lange et al., 1984; Orchard and Lange,1986), which still produce rhythmic myogenic contractionswhen isolated from the central nervous system. Lesion 2leaves the PVG intact and does not affect the contractionrate of the proventriculus. Together with the effects oflesion 1 this implies that proventriculus contractions aremodulated by a yet unknown neuronal or humoral factorreleased from the PVG, but not the HCG. It was shown forCalliphora larvae that fibres immunoreactive againstcorazonin (cockroach cardio active peptide) and serotoninproject along the recurrent- and proventricular nerve to thePVG (Cantera, 1988; Cantera et al., 1994). So possibly theapplied lesions affect a modulatory pathway, which servesto adapt the foregut activity to the respective nutritionalrequirements. Alternatively, the foregut activity is possiblymodulated by sensory input from the midgut. Similar toadult blowflies (Gelperin, 1971a) and crickets (Mohl, 1972)a feedback loop might exist over which an empty midgutincreases foregut activity to accelerate food transport. Theproventricular- and recurrent nerves provide an evidentpathway for this loop. Opening the loop (lesion 1 and 2)possibly mimic the effect of a full midgut and thus reducethe activity of the foregut. A second possibility would bethat so far unidentified local myogenic pacemaker regionsregulate the activity of the oesophagus and the crop ofCalliphora larvae. Such regions are described for theforegut of adult cockroaches (Cook et al., 1969). We canso far not solve the question how exactly the movements ofthe oesophagus, crop and proventriculus are generated andcontrolled. We observed the described movements oncompletely isolated crops, oesophagi and proventriculi.Therefore, we propose that the movements are myogenic

and that their frequency is modulated by the nervoussystem.

4.5. Efferent (motor) pathways

The insect SNS generates the motor patterns ofpharyngeal muscles necessary for feeding (Ayali, 2004;Ayali et al., 2002; Miles and Booker, 1994, 1998). Electricalstimulation of the AN of Calliphora demonstrates that thisnerve contains fibres that innervate the CDMs. The fibresprobably project through the AN into the FCs, pass theFG and innervate the CDM via the FN. This correspondsto the occurrence of synchronous bursts of actionpotentials in the ANs, which correlate with postsynapticpotentials on the CDM. Successive transections of the ANsdemonstrate that a muscle bundle is innervated by fibresfrom both ANs. The variation in PSP amplitude on theCDMs can be caused by different motor units whosethresholds are so close together that the given stimulus canactivate more than one unit. To gain information on thenumber and properties of putative motor units, experi-ments are in progress with varying stimulus amplitudes, asperformed on locust oviduct muscles (Orchard and Lange,1986), or mandibular closer muscles of larval Manduca

sexta (Griss, 1990). The extracellular recordings from theAN support the hypothesis of polyneural innervation ofthe CDM. Each burst contains action potentials of at leastthree distinct amplitudes, which resembles the activationpattern of insect skeletal muscles by ‘‘fast’’ and ‘‘slow’’motor neurons (Burrows, 1996).The motor programme for food ingestion recorded from

the AN resembles the rhythmic neural activity underlyingfeeding in Manduca sexta (Miles and Booker, 1994, 1998)and Locusta (Ayali and Zilberstein, 2004; Ayali et al., 2002;Zilberstein and Ayali, 2002). A fundamental difference inneural organisation appears to exist between Calliphora

larvae and the preparations named above. In both,Manduca and Locusta motor neurons driving foregutmuscle contractions are located in the FG (Ayali andZilberstein, 2004; Miles and Booker, 1994, 1998; Zilber-stein and Ayali, 2002). In Calliphora, all motor activity inthe AN is abolished after cutting the nerve anterior to thebrain. This strongly suggests that in Calliphora larvae themotor neurons of the CDM are not located in the FG but,as in adult Drosophila, in the tritrocerebral part of thebrain (Rajashekhar and Singh, 1994; Tissot et al., 1997).The motor axons projecting through one AN innervate

both, ipsilateral and contralateral, CDM bundles, asdemonstrated by subsequent transection of the ANs. Toour knowledge insect motor neurons generally projectthrough one nerve to innervate muscles unilaterally(Burrows, 1996). Therefore, the innervation pattern ofCalliphora CDM musculature appears to be rather excep-tional. We do not have any information on developmentalor functional aspects of this innervation pattern, but itmight serve to ensure synchronous contractions of musclebundles on both sides for optimised food ingestion.

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4.6. Afferent (sensory) pathways

Ten minutes after hungry larvae are transferred to freshmeat the crop is filled with food. To avoid hyperphagia, thefeeding behaviour must be adjusted to the filling of thecrop. Experiments in which the crop volume was artificiallymanipulated demonstrate that (so far unidentified) stretchreceptors in the crop can provide the necessary sensoryfeedback. Artificial crop inflation results in increasedneural activity in the recurrent nerve. Comparable sensoryfeedback exists in adult blowflies (Dethier and Gelperin,1967; Gelperin, 1967, 1971a, b, 1972). Double recordingsfrom the recurrent nerve and the proventricular nerve showthat the action potentials travel toward the proventriculus.Thus, possibly stretch receptors in the crop contribute tothe putative feedback loop discussed above, whichregulates foregut movements. It should be expected thatthe central nervous system integrate information on cropvolume to adapt the ongoing feeding behaviour. Anapparent pathway for sensory information from the cropto the brain would be via the recurrent nerve, the FCs andthe ANs. Surprisingly, the neural activity in the AN doesnot depend on crop volume. We conclude that the putativeinfluence of crop volume on feeding behaviour is moresubtle than a direct inhibition of the motor neurons in thebrain. As in adult blowflies feeding behaviour might beregulated by osmotic pressure of the food and haemolymph(Gelperin, 1966).

The fine structure of the DO of house fly (Musca

domesticus) larva is described in great detail (Chu andAxtell, 1971). It consists of a dome-shaped structure placedin a cylindrical socket on the cephalic lobe. The DO isinnervated by 35–41 neurons, which form a ganglion withinthe cephalic lobe. The dome is innervated by seven axonbundles of 21 bipolar neurons. The scolopidia present inthe DO are propably stress receptors (Chu-Wang andAxtell, 1972). For Drosophila, a rich literature is availableabout the DOs and the role they play in olfaction (Cobb,1999; Gendre et al., 2004; Heimbeck et al., 1999; Jurgenset al., 1986; Opplinger et al., 2000; Python and Stocker,2002a, b; Ramaekers et al., 2005; Tissot et al., 1997). Ourexperiments confirm the findings of (Chu-Wang and Axtell,1972) and demonstrate a mechanosensory function of theDOs. A clear sensory response in the AN is evoked by adislocation of the dome by only 10 mm, so probably evensmaller mechanical stimuli can be detected by the organs.During feeding, the CPS is telescopically extended and theDOs located on the cephalic lobes are exposed to thesurrounding substrate, which certainly causes a sensoryresponse. Optimised temporal correlation of CDM con-traction and extension of the CPS could be mediated bysensory input from the DOs to the SNS.

The results of this paper provide a first insight to theanatomy and function of the larval Calliphora SNS.Further investigations will concentrate on the neuronalnetworks involved in rhythmic feeding behaviour and theinfluence of sensory input on these circuits.

Acknowledgements

We want to thank S. Niederegger, M. Gephardt and M.Hofmann their comments on the manuscript. B. Usai forfilming the foregut movements, and V. Monar for herdrawing of the Calliphora larva.

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