Symmetrically Organized Dorsal Unpaired Median (Dum) Neurones and Flash Control in the Male Firefly, Photuris Versicolor by Thomas A

r exp. Biol. (1981), 93. I33"i47 133 Hbt/i 8 figures Printed in Great Britain SYMMETRICALLY ORGANIZED DORSAL UNPAIRED MEDIAN (DUM) NEURONES AND FLASH CONTROL IN THE MALE FIREFLY, PHOTURIS VERSICOLOR BY THOMAS A. CHRISTENSEN AND ALBERT D. CARLSON Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York 11794 USA (Received 1 October 1980) SUMMARY 1. Male fireflies of the species Photuris versicolor produce a species-typical triple-pulsed flash which is used as a courtship signal. The neural anatomy was examined to determine if this complex behaviour could be attributed to the organization within the central nervous system. 2. The lantern is innervated primarily by the two most posterior abdominal ganglia. Bilateral roots from these ganglia form a symmetrical pattern of innervation to both sides of the lantern tissue. With minor exceptions, this pattern is similar to that described for other firefly species. 3. The neural organization within the lantern ganglia was determined by back-filling the roots with cobalt or Lucifer Yellow CH, and then examining the ganglia in whole mount. Clusters of three or four large dorsal unpaired median (DUM) neurone somata, each sending bilateral processes out of the lantern roots, were found in both lantern ganglia. 4. The DUM neurone axons bifurcate several times and ramify throughout the dorsal surface of the lantern tissue. More than one DUM neurone may innervate a particular region of photogenic tissue. 5. When dye was back-filled into peripheral branches of the lantern roots that do not innervate photogenic tissue, no DUM somata were stained. Instead, the fibres that filled carried the dye anteriorly up the nerve cord through the ipsilateral connective. No fibres were observed to cross the ganglion midline or exit from the contralateral root, nor were any fibres stained in the contralateral connectives. 6. DUM neurones within the lantern ganglia have resting potentials between 30 and 45 mv and they exhibit multiple, as well as single-peaked spontaneous action potentials. The presence of multiple spikes might reflect the special bilateral morphology of these neurones. 7. The lantern nervous system is organized in an arrangement capable of synchronizing the excitation of all the lantern photocytes. This neural organization could aid in the control of the complex flash pattern displayed by male Photuris versicolor fireflies. INTRODUCTION The firefly flash is a rapid burst of light used as a courtship signal, and each species firefly produces a unique flash exchange pattern (Lloyd, 1966). Male Photuris I 'eflies, in particular, produce complex flashes by rapid variations of flash intensity 134 T. A. CHRISTENSEN AND A. D. CARLSON B Fig. i (A). Relationship between electrical activity in the lantern tissue and the dynamics of a P. verticolor male spontaneous triple-pulsed flash. Upper trace: photomultiplier output of light emission. Lower trace: spike activity recorded via external electrodes in the anterior lantern segment. Temp: 22 °C. Time marker: 200 ms. (B) Gross innervation of the lantern (dorsal view). The two large lantern organs (shaded) completely cover the sternitea of the 6th and 7th abdominal segments. The lantern tissue is innervated by paired roots from the 5 th, 6th and 7th abdominal ganglia. These roots break up into a network of smaller fibres (not illustrated) that densely innervate all the photogenic tissue. Note also that intersegmental tracts link adjacent roots in the periphery. The four genital roots from A 7 are cut short in this diagram. Scale marker: 1 mm. (Barber, 1951), and these modulated flashes are recognized by prospective mates of the same species during courtship (Nelson, Carlson & Copeland, 1975; Zorn & Carlson, 1978). The P. versicolor male typically emits a triple-pulsed flash which appears as a twinkle to the human observer (Fig. 1 A). The rhythmic flash patterns of many fireflies, including P. versicolor, are believed to be controlled by a neural 'pacemaker* within the animal's brain(Case & Buck, 1963; Bagnoli et al. 1976). Bursts of action potentials travel down the segmental nerve cord and by some means activate the photocytes (light-producing cells) of the lantern. These action potentials can be recorded by external electrodes in the lantern tissue (Case & Buck, 1963; Fig. 1 A). In P. versicolor males, the neural volley activates both lantern segments simultaneously, resulting in three rapid pulses of light from the photogenic tissue. Although Chang (1956), and later Buck & Case (1961) demon- strated that the adult firefly lantern behaves like a typical neuroeffector such as striated muscle, we still do not fully understand how neural activity initiates luminescence in the photocytes (Case & Strause, 1978), which are actually derived from fat cells (Hess, 1922). The gross neural anatomy of the lantern was originally described by Hanson (1962). The lantern, which occupies the sternites of the sixth and seventh abdominal segments, is innervated by nerve roots from the fifth, sixth and seventh abdominal ganglia (A 5, A 6 and A 7, respectively), the latter two ganglia lying dorsal to the anterior lantern segment (Fig. 1B). Hanson (1962) showed that it was possible to deganglionate the posterior lantern segment by severing the connexions with its more anterior ganglion, A 7. The adult lantern ultrastructure was first described for a Photinus species by Beaim & Anderson (1955), and later for Photuris by Kluss (1958) and Smith (1963). DUM neurones and flash control in the firefly 135 Prganized into a ventral photogenic layer and an overlying reflector layer. Trachea and nerves plunge ventrally into the photogenic tissue forming a uniform array of tracheal cylinders. Tracheal processes, enclosed in tracheolar cells, project horizontally away from the tracheal cylinders, through specialized, mitochondria-filled tracheal end cells, and eventually reach the photocytes which are situated in a rosette pattern around the tracheal cylinders. Nerve axons split away from their surrounding sheath cells and terminate in pad-like endings between the tracheal end cells and tracheolar cells. Synaptic specializations can be seen between the vesicle-filled nerve endings and tracheolar cells, and it is believed that these cells transmit the excitation to the photocytes (Case & Strause, 1978). Several lines of evidence indicate that the phenylethylamine octopamine could function as a neurotransmitter in the lantern. It has been shown that this amine is extremely effective in eliciting lantern luminescence (Carlson, 19686), and that substantial amounts are found in the lantern segments (Robertson & Carlson, 1976). More recently, octopamine has been found to activate an octopamine-sensitive adenylate cyclase in the lantern which catalyses the production of cyclic AMP (Nathanson, 1979). It is suggested that activation of the photocyte-end-cell complex could occur through the actions of this second messenger, but the details of activation remain unresolved. From this description it is clear that we understand a great deal about the neural and physiological control of firefly flashing, but the anatomical organization within the central nervous system, through which the triggering bursts from the brain pass to the lantern, remains unexplored. In this article we report the discovery of distinct popu- lations of large dorsal unpaired median (DUM) neurones within the lantern ganglia that appear to perform a key function in conducting the neural bursts from the brain to the photocytes of the lantern. Moreover, these neurones are organized to ensure the synchronous activation of the thousands of photocytes in both lantern segments. To further define the functional significance of these neurones, we have monitored their spike activity and identified them anatomically using intracellular recording and dye- marking techniques. The results presented here match the expected results, given the geometry of the neurones and their proposed function. In short, these cells appear to be both physiologocally and anatomically suited to an important role in the production of light by the photocytes. A preliminary report of these results has appeared elsewhere (Christensen, 1980). METHODS Organisms Adult male fireflies (Photuris versicolor) were obtained from a tree-lined grassy field near Stony Brook, Long Island, New York. Males displaying their species-specific triple-pulsed flash were lured out of the trees with a single flash from a flashlight pointed into the grass, which served to mimic the typical female response to the male's flash. After capture, the fireflies were kept in glass or plastic containers at room tem- perature with moist paper-towelling and grass. All were dissected within a few days after capture. 136 T. A. CHRISTENSEN AND A. D. CARLSON Anatomical methods The gross morphology of the lantern nervous system was examined by two methods. To locate the destinations of the lantern ganglia roots, the abdominal dorsal cuticle was removed and the exposed ventral nerve cord and lantern were stained with methylene blue dye (1%, w/w, in saline; Hanson, 1962). The finer peripheral nerve processes were traced by back-filling one lantern ganglion root with cobaltous chloride (6%, w/w, in distilled water, containing o-i% bovine serum albumin), and allowing the cobalt to be carried into the ganglion and out again through the contralateral nerve root to the photogenic tissue. Subsequently, the preparation was rinsed with ammo- nium sulphide which

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