From bioassays to Drosophila genetics: strategies for characterizing an essential insect neurohormone, bursicon.

We describe the molecular analysis and cellular expression of the insect peptide neurohormone, bursicon. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, we determined the molecular weight of bursicon from different insects to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, we successfully searched the Drosophila genome project for the gene encoding bursicon. With Drosophila as a tool, we can now verify the function of the sequence using transgenic flies. Sequence comparisons also allowed us to verify that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution.

Physiological characterisation of antennal mechanosensory descending interneurons in an insect (Gryllus bimaculatus, Gryllus campestris) brain.

We investigated five different descending brain interneurons with dendritic arborizations in the deutocerebrum in the crickets Gryllus bimaculatus and G. campestris. These interneurones convey specific antennal mechanosensory information to the ventral nerve cord and all responded to forced antennal movements. These interneurones coded for velocity and showed preferences for distinct sectors of the total range of antennal movements. Their axons descended into the posterior connective either ipsilateral or contralateral to the cell body. Electrical stimulation of sensory nerves indicated that the interneurons received input from different afferents of the two antennal base segments. One interneuron had a particularly large axon with a conduction velocity of 4.4 ms(-1). This was the only one of the five interneurons that also received visual input. Its activity was reduced during voluntary antennal movements. The reduction in activity occurred even after de-efferentation of the antenna, indicating that it had a central origin. Although we do not have experimental evidence for behavioural roles for the descending antennal mechanosensory interneurons, the properties described here suggest an involvement in the perception of objects in the path of the cricket.

Glutamate-like immunoreactivity marks compartments of the mushroom bodies in the brain of the cricket.

In the mushroom bodies of the brain of the cricket Gryllus bimaculatus, the distribution of glutamate-like immunoreactivity is shown by using several immunocytochemical staining protocols and confocal and conventional microscopy. Glutamate-like staining of intrinsic cells of mushroom bodies (Kenyon cells), their axons and projections, is demonstrated for the first time. Two types of Kenyon cells constituting distinct, separated populations within the perikaryal layer and in prominent neuropilar subcompartments exhibit strong (type III cells) or medium (type II cells) glutamate-like immunoreactivity, whereas the small neurons of a central population (type I cells) lack staining above background. Type III Kenyon cells display a strong immunoreactivity similarly found in some giant neurons and in identified antennal motorneurons by using glutamate as an excitatory transmitter, indicating that also distinct populations of the Kenyon cells use glutamate as a putative transmitter. The pattern of glutamate-like immunoreactivity in the mushroom bodies and in other parts of the brain is different from gamma-aminobutyric acid (GABA)-like immunoreactivity (investigated for comparison). GABA-like immunostaining is particularly prominent in the mushroom body calyces where Kenyon cells have their dendritic branchings. Differences in glutamate-like immunostaining in Kenyon cell subpopulations, together with differences in their arborization and axonal projection patterns, indicate a functional diversity of these neurons.

Antisera against Periplaneta americana Cu,Zn-superoxide dismutase (SOD): separation of the neurohormone bursicon from SOD, and immunodetection of SOD in the central nervous system.

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon.

The antennal motor system of crickets: proctolin in slow and fast motoneurons as revealed by double labelling.

This study describes proctolin-like immunoreactivity (PLI) of identified antennal motoneurons in the brain of adult crickets (Gryllus bimaculatus). The motoneurons were first backfilled with the fluorescent dye Lucifer Yellow and then immunohistochemically labelled with an antibody against proctolin. Altogether 14 of the 17 excitatory antennal motoneurons, including physiologically fast and slow types, showed PLI. The only common inhibitor consistently demonstrated a weak positive PLI. PLI was also present in the dendritic arborizations and varicosities of motor axons in the intrinsic antennal muscles. Densitometric measurements of motoneuron somata showed significant differences in the intensity of PLI in different types of antennal motoneurons, suggesting that antennal motoneurons produce different amounts of proctolin. Identical motoneuron somata display a large variance of PLI intensities in different brains. This observation may indicate up- and down-regulation of proctolin in individual crickets.

Morphology of antennal motoneurons in the brains of two crickets, Gryllus bimaculatus and Gryllus campestris.

The morphology of the antennal muscles of two cricket species, Gryllus campestris and G. bimaculatus, and their innervation are described. The motoneurons innervating the five tentorio-scapal muscles M4 and M5 and the two scapo-pedicellar muscles M6 and M7 were stained with cobalt chloride introduced via the cut axonal endings in the muscle. The seven antennal muscles are innervated by a total of 17 excitatory motoneurons and one common inhibitory neuron. These neurons branch in the dorsal neuropil of the deuto- and tritocerebrum. No difference in the morphology of the motoneurons between the two species was evident. Two dorsal-unpaired-medial (DUM) neurons located in the suboesophageal ganglion also innervate the antennal muscles. Intracellular recordings of some motoneurons combined with Lucifer Yellow injections corroborated the motoneuron morphology obtained by cobalt backfilling from the muscles. The recordings showed that the motoneurons are either of the fast or the slow type.

On-line analysis of rapid motion with a microcomputer.

A software package is described, which uses an Apple IIe computer with a digitizing board (Digisector 65) for analysing rapid motions of appendages of small insects from video images. Every 20 ms the program simultaneously analyses the position of two moving appendages after a background correction and the externally applied trigger stimulus. The data may be plotted in high-resolution plots with a matrix printer and evaluated with statistical methods.

A comparative study of neck muscle motor neurons in a cricket and a locust.

The gross morphology of the neck muscles of a cricket (Gryllus campestris) and their innervation are described and compared with a locust (Schistocerca gregaria). The motor neurons innervating the neck muscles were stained in crickets and locusts with cobalt chloride introduced via the nerve endings in the muscle. The two species show overall similarities, not only in position of the neck motor neurons in suboesophageal, prothoracic, and mesothoracic ganglia but also in motor neuron morphology. However, muscle 60 in the cricket is innervated by a unique motor neuron with its axon in prothoracic nerve 3, instead of sharing motor neurons in suboesophageal nerve 8 and mesothoracic nerve 1 with muscle 59, as in locust. Muscle 62 has the same attachments and innervation with similar motor neurons in cricket and locust but a different mechanical function in the two species. The findings are discussed with respect to possible segmental homologies and to the origins of the muscles as either dorso-ventral or longitudinal. As several muscles share the same motor neurons, we suggest that neck muscle function be described in terms of "behavioural units of action."

The ultrastructure of campaniform sensilla on the eye of the cricket, Gryllus campestris.

The structure of the campaniform sensilla of the cricket eye was investigated by light and electron microscopy. Each sensillum is innervated by a single bipolar neuron. Its axon extends through the retina into a side-branch of the nervus tegumentarius. The dendrite extends through a cuticular channel to the surface of the cornea. The distal part of the dendrite, the sensory process, contains a tubular body and is attached to a cuticular cap which is obliquely inserted into the exocuticle between the corneal lenslets. Some particular structural features as well as the function of the campaniform sensillum of the cricket eye are discussed.

Interommatidial hair receptor axons extending into the ventral nerve cord in the cricket Gryllus campestris.

In the cricket, Gryllus campestris, a branch of the nervus tegumentarius runs to the distal part of the optic lobe. This branch contains the axons of interommatidial hair receptors. The axon terminations extend forward into the trito- and deutocerebrum, and into the subesophageal- and prothoracic ganglia as shown with the cobalt sulfide staining technique. The possible relevance of these connections is discussed.

Cobalt sulphide staining of optic fibres in the brain of the cricket, Gryllus campestris.

Neuronal projections from one optic lobe to other parts of the brain were stained in the cricket Gryllus campestris using the cobalt sulphide technique and Timm's sulphide-silver method. The results are: Four tracts directly connect the medulla with the lobula and medulla of the contralateral optic lobe. Direct medullar projections end mainly in the non-glomerular neuropile of the protocerebrum, but also penetrate the calyx of the mushroom bodies, pons and central body in small numbers. A few somata which send fibres into the medulla lie in the pars intercerebralis, in the protocerebrum ventral to the opposite beta-lobe, the outer margin of the medulla of the contralateral optic lobe and between deuto- and tritocerebrum. The anatomical and physiological relevance of the stained connections is discussed.

Adaptations of the intertidal midge Clunio to arctic conditions.

1. The arctic population of Clunio marinus in Tromsö (Norway) (69°39'N) shows a tidal periodicity of emergence (period: 12.4 hours). Emergence occurs between the times of high and low tide. 2. A semilunar periodicity of pupation could not be detected. 3. In accordance with the tidal periodicity of emergence and reproduction, the habitat is situated in the midlittoral zone (area between the high and low water levels of neap tides). High densities of pupation are found in a sandy mud flat. 4. The Tromsö population is compared with European populations in temperate latitudes, which are characterized by a semilunar periodicity of emergence, and a habitat around the infralittoral fringe of the midlittoral zone. The different adaptations of the populations in biological rhythms and preferred habitat are discussed with regard to the geographic differences in photoperiodic conditions.