Flash Communication in Fireflies
Transcription
Flash Communication in Fireflies
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We work with the scholarly community to preserve their work and the materials they rely upon, and to build a common research platform that promotes the discovery and use of these resources. For more information about JSTOR, please contact [email protected]. The University of Chicago Press is collaborating with JSTOR to digitize, preserve and extend access to The Quarterly Review of Biology. http://www.jstor.org VOLUME 60, THE No. 4 DECEMBER QUARTERLY REVIEW of B IOLOGY COMMUNICATION IN INSECTS I. FLASH COMMUNICATION IN FIREFLIES ALBERT D. CARLSON & Behavior,State Universityof New York, Department of Neurobiology Stony Brook, New York11794 JONATHAN COPELAND * Departmentof Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201 USA ABSTRACT Thefireflyflash is a signal in a courtshipsystembasedupontimingpatternscontrolled by thebrain.Courtshipflash codessupplybothspeciesandsexidentification andrelyonprecisetimto preserveinformation. ing and stereotypy of oneor a few temporal parameters Theflashcontrolsystemis physiologically complex.It involvesrandom physiologicalfluctuationsthattendto introduce andthusto degrade transvariabilityintocommunication information thephysiological basisofflashcommunication it is therefore essentialtoestablish fer. To understand thelimitsoffluctuationwithinwhichtheentiresystemmustoperate.Thephysiological processes thatareso variableinvolvesensoryreception, centralintegration, and theoutputof an unusual neuroeffector (thefireflylantern).Forexample,thepatterngenerators offirefliesmustbeprimed to operatewith sufficientprecision. Insufficientlyprimed animals produceflashes of abnormal kinetics and timing. Flashing behaviorof fireflies is greatly affected by the animal's state of arousal; quiescentfireflies are inducedtoflash with difficulty, whereasexcitedanimals produce rapid and variedluminescentemissions. Also, thefirefly's visual apparatusis highly specialized for the receptionoffast transientflashes, and its ability toprocessothertypesof photic information remainsunknown. Finally, andperhapsmostsignificantly, theflash patterngeneratorsoffireflies can be entrainedby incomingflashes to produce, in females, responsesof appropriatedelay; and in males, flash synchrony. Lloyd (1981b) has proposedthat aggressivemimicry by Photuris femmes fatales has had a great evolutionaryimpact on theflash codesof preyfirefly species. This mimicrymay represent a simple entrainmentof Photuris females to a wide arrayofflash patterns. Theprecisionofflash behaviorshould be measuredin females of known physiological state and previousflash history in orderto assess the effect of mimicry on firefly courtship behavior. * Present address: Dept. of Biology, SwarthmoreCollege, Swarthmore,Pennsylvania 19081 USA. ? 1985 by the Stony Brook Foundation, Inc. All rights reserved. 415 1985 416 THE QUARTERLY REVIEW OF BIOLOGY INTRODUCTION HE FLASH of a firefly, a brillant burst of light lasting fractions of a second, is a signal in a courtship and mating communication system in which species and sexual identity are conferred by flash timing parameters (McDermott, 1910-1917; Buck, 1937; Barber, 1951;Lloyd, 1966a). The flash code involved in courtship and mating behavior is both stereotyped and interactive. In the simplest and least ambiguous case, something that rarely occurs in the field and must usually be achieved through controlled laboratory or field studies, an isolated pair of individuals produces stereotyped flash patterns. Male and female thus act as both sender and receiver in "typical"firefly flash communication, and little variability in signaling is to be seen. Firefly communication, however, does show some variability, and such variability (noise) may enter at any stage. Such stages involve sensory reception, central processing of the photic signals, and motor output to a highly unusual effector organ, the firefly lantern. This variability may be quantified and its limits defined. Additional sources of variability also exist the recognition of which is critical to an understanding of firefly communication: (1) small changes or nuances in timing patterns may occur during the later stages of firefly courtship; (2) there may be changes in the central excitatory state of either organism; and (3) previous flash and mating history result in variability. The stereotyped nature of firefly flash communication is thus somewhat illusory. Fixity of pattern exists to a certain extent in both laboratory and field, but upon arrival in the field an observer is struck with the cacophony of flashes with which a firefly must contend. Recently there have developed two approaches to the analysis of variability seen in the field: an adaptationist approach and a neuroethological approach. In the adaptationist approach, functions are assigned to all flashes observed, and the variability seen is thought to be maintained in the population by sexual selection (Lloyd, 1969a). Not all flashes, then, are considered to be courtship T [VOLUME 60 flashes, in the simplistic sense indicated above. Fireflies must also contend with numerous competing deceitful flash strategies, such as rivalry flashes, designed to win females in courship competition. Thus, injected flashes (Lloyd, 198 la) and transvestite flashes (Lloyd, 1984) have been described to explain some of the variability seen in the field. In addition, some flashes seen in the field are only indirectly involved in sexual selection for they are actually used in predation. Flash mimicry by both male (Lloyd, 1980) and female (Lloyd, 1965) fireflies has been hypothesized to have evolved in obtaining food or, indirectly, in the search for a mate. Still other flashes are thought to be involved in behaviors other then courting. For example, landing flashes are supposed to light up a landing site (Lloyd, 1968; Papi, 1969); some flashes are said to aid the female in finding a suitable egg-laying site (Kaufmann, 1965); or aerial predators ("hawkers") home in on flashing targets (Lloyd and Wing, 1983). The second, or neuroethological approach, suggests that (1) the firefly's nervous system is designed to produce flashes in stereotyped timing patterns; (2) the variability seen in the field can be largely explained by definable and measurable physiological fluctuations; (3) the firefly's state of arousal and its previous flash history are critically important in understanding the meaning of particular flashes; and (4) most flashes involved in communication can be described and modelled according to certain simple physiological rules. Fireflies present a nearly ideal group of insects for neurobiological or behavioral study of animal communication (Lloyd, 1983), for a number of reasons. First, not only is firefly communication dynamic and interactive, both males and females exchanging information rapidly, but also the signals can be observed and recorded at a distance without affecting the interaction. The observer can, furthermore, easily participate in the exchange by means of simple equipment such as a flashlight. In addition, the rationales and results of the communication paradigm are usually obvious. Second, the communication system is amenable to a degree of neu- 1985] DECEMBER FLASH COMMUNICATION 417 20 16 _ 1st BURST N=75 12 8- z 4-) I1 1I z u I 1 1 A 20- , 0 oL 0 16- 2nd 12 N=74 BURST idIL Th4I6 8- z r LU 4 D- 20 3rd BURST N=59 16_ 2 84_ 8 12 16 20 24 NUMBER OF SPIKES PER BURST FIG. 1. RELATIONSHIP BETWEEN NEURAL ACTIVITY RECORDED IN THE LANTERN TISSUE AND THE TRIPLE-PULSED COURTSHIP FLASH OF A Photurisversicolor MALE (A) Photomultiplieroutput (upper trace) and action potential volleys (lower trace) recorded from the anterior segment of a lantern during two courtship flashes of a restrained male. The average interval between the first and second flash pulses was 243.9 + 8.67 (S.D.) msec (N=98); between the second and third flash pulses it was 236.7 + 9.04 (S.D.) msec (N=86). The bar equals 100 msec. (B) The variation in number of spikesper burst in the three volleys that trigger the pulses of the courtship flash. No consistent relationship was observed between spike numbers and flash pulse intensities. Temperature, 210C. rophysiological analysis, because fireflies of some species will continue their courtship behavior when restrained or even in dissected preparations (Fig. 1). Third, each species of firefly produces a different courtship code (Lloyd, 1966a), differences that make comparative studies possible. Fourth, the flash patterns are clearly under the control of the firefly's brain (Case and Buck, 1963; Buck, Case, and Hanson, 1963) and therefore rep- resent patterns of activity that can provide insight into the insect's brain function. All of these advantages make firefly courtship a powerful communication system that lends itself to the precise analysis of behavior. In this review we first provide a brief description of the natural history of fireflies. To demonstrate the potential complexity of courtship communication, the courtship of Photinusmacdermottifireflies is described in de- THE QUARTERLY REVIEW OF BIOLOGY 418 tail. Next, the physiological basis of flash control is described. This section included a description of lantern anatomy and innervation, of flash production, of the sensory reception of flashes, of the central programming of flash behavior, and of synchronous flashing as an example of the role of pacemakers in flash timing, and an analysis of flash entrainment by Photuris versicolor females. This section is completed by summary of the physiological processes that influence and control flash behavior. In the next section, this physiological background is used to analyze predatory mimicry by female Photuris fireflies. The importance of understanding the physiological limits inherent in firefly flash communication before assigning putative functions to flash behaviors observed in the field is discussed. THE NORTH NATURAL HISTORY AMERICAN OF FIREFLIES Fireflies are luminescent beetles of the family Lampyridae (McDermott, 1964). The description that follows is confined to the two principal genera of north American fireflies: the yellow-green-flashing Photinus, the taxonomy of which has been studied by Green (1956) and by Lloyd (1966a, b, 1969b); and the green-flashing Photuris, the taxonomy of which is less well known (Barber, 195 1; Lloyd, 1969b). The life cycle of a firefly is typical of a coleopteran. It begins with the egg stage, followed by an overwintering larval stage of a number of instars, then by pupation in the spring, and eventual eclosion as an adult, usually in June or July. Photinus larvae are only rarely found on the surface, as they spend most of their lives under the leaf litter or in the soil. Photurislarvae, in contrast, are found in large numbers at night on the surface of the ground, in moist areas in late spring and early autumn. They apparently require two years for development (Williams, 1917; Hess, 1922; McDermott, 1958; McLean, Buck and Hanson, 1972). This larval life stage is carnivorous. The larvae feed on other insect larvae, worms, and molluscs, which they immobilize by injecting poison into them through their curved mandibles (Fabre, 1919; Schwalb, 1961; Copeland, 1980). [VOLUME 60 The larval firefly possesses tiny, paired lanterns on the sternite of the eighth abdominal segment. These lanterns differ significantly in structure (Buck, 1948; Peterson, 1970) and in biochemistry (Strause and DeLuca, 1981) from the adult's lantern, which occupies all or a portion of the sixth and seventh abdominal sternites. The larval lantern is incapable of producing a flash. Instead, the insect produces a glow that waxes and wanes over a period of seconds. The role of the larval lantern has been the object of considerable speculation (Sivinski, 1981). One possible function is as an aposematic signal to warn nocturnal predators of the insect's unpalatability (Carlson and Copeland, 1978). This interpretation is suggested by two observations: (1) that Photuris larvae luminescence immediately upon being touched or handled; and (2) that they exude a liquid which appears to be noxious to mammals such as mice (Carlson and Copeland, 1978). Finally, the fact that adult fireflies are unpalatable to a number of potential predators (Lloyd, 1973a; Vernon, 1983) also suggests aposematic signalling. Many vertebrates, such as thrushes (Eisner, Wiemer, Haynes, and Meinwald, 1978) and the big brown bat (Vernon, 1983), reject adult fireflies promptly or avoid them entirely. Adults of some species exude a liquid that appears to be noxious to ants and lizards (Williams, 1917; Blum and Sannasi, 1974). Their unpalatability is due, in part, to lucibufagins (Eisner et al. 1978), so-called because these substances resemble bufodienolides, the cardiotonic agents present in toad skin. The Photuris larva overwinters within a pupal cell constructed of mudballs (Hess, 1920; McLean, Buck, and Hanson, 1972). In the second spring it pupates and develops the adult lantern on the sixth and seventh abdominal sternites. Initially, the lantern can only produce a glow, which eventually can be quenched in scattered areas of the lantern. Slowly the ability to produce a flash arises (Carlson, 1969), the process being still incomplete after the eclosion and emergence of the adult (Strause, DeLuca, and Case, 1979). During this period the larval lanterns usually degenerate and disappear. Harvey and Hall (1929) excised the larval lanterns DECEMBER FLASH COMMUNICATION 1985] without any effect on development of the adult lantern, and thus established that the former is not a precursor of the latter. THE COURTSHIP OF Potinus macdermottiFIREFLIES The Nearctic Photinus fireflies include several dozen species (Green, 1956; Lloyd, 1966a). They communicate by one of two basic flash codes. The simplest code is typified by P. pyralis, the most widespread American firefly. It involves a series of consecutive flashes emitted by the male at intervals of about 6 seconds at 23?C and a single flash answer by the female approximately 2 seconds after each male flash (Buck, 1937). The other courtship flash code is emitted by P. consanguineus, P. greeni, and P. macdermottifireflies, members of the so-called consanguineus group (Lloyd, 1966a). In these, the male emits courtship flashes in pairs, with a fixed interval between the two flashes of the courtship pair, and a variable period between the pairs. The female responds after the second flash of each pair, twisting her lantern in the male's direction. The courtship of P. greeni has been studied intensively by Buck and Buck (1972). All flash-timing parameters, including flash duration and frequency, are affected by temperature (Buck, 1937; Edmunds, 1963; Carlson, Copeland, Raderman, and Bulloch, 1976; Carlson, 1981). Unless otherwise specified here, flash periods and latencies are given for approximately 20?C. Photinus macdermottimales emit a pair of courtship flashes of approximately a 2.3second period and receive answers from the perched female about 1.5 second after the second flash (Lloyd, 1966b, 1969b; Carlson et al., 1976, 1977). If a male approaches a female through the grass, he will climb a blade, stop, emit a pair of flashes, and receive an answer from the stationary female. He will then move to the top of another blade of grass before emitting his next courtship pair of flashes. If a male approaches a female from the air, he will hover in flight in front of the female and will fly upward between his courtship flashes. Upon receiving an answer, he will dip and climb again while flashing. These courtship flashes become extremely dim as courtship proceeds, so that the possi- 419 bility of detection by other males is reduced. This behavior is similar to that found in orthopteran males, in which pair-forming signals are loud, but those emitted by males near females are characteristically muted (Otte, 1972). Flying Photinus macdermottimales who have not located a female produce strings of rhythmically repeated flashes approximately onehalf second longer in flash period (i.e., 2.6 sec.) than courtship flashes (Carlson et al., 1976). This seemingly insignificant difference in timing produces a change in female behavior. The longer interval between these flashes induces females to respond after each flash instead of answering every alternate flash, like females replying to flashes in the courtship rhythm. Perhaps, by receiving answers to consecutive flashes, the flying male can obtain information from the female more rapidly, and this signal enables him to locate her more precisely. The paired courtship flashes, emitted once the male has located a female, are apparently designed, in part, to establish that the answers are coming from a conspecific female. A courting male might be expected to ignore flashes that arrive between his first and second courtship flashes because such answers are not appropriate to the basic firefly code. However, experiments with courting males have revealed that this assumption is 1978). wrong (Carlson and Copeland, Flashes that arrive within one second after the first courtship flash cause the male to delay his second courtship flash by up to 300 msec. Perhaps the male interprets the premature answer of the female as a flash from another male and, by delaying his second flash, attempts to synchronize more closely with the intruding male's second flash. If the intruding flash arrives between 1 and 1.5 seconds after the first flash, the courting male cancels his second flash and initiates another pair of courtship flashes. He may interpret this flash as coming from the female because it is properly timed, even though it follows the wrong flash. Flashes that arrive later than 1.5 seconds after the courting male's first flash usually have no effect on his second flash, probably because the male is already committed to emit that flash. A rival male that happens upon a court- 420 THE QUARTERLY REVIEW OF BIOLOGY ship in progress adopts one of two behaviors designed to compete for the female's attention. He attempts either to synchronize his flash with the courting maie's second flash, or he flashes within a second after the female's response (Lloyd, 1981a,b; Carlson and Copeland, 1983). By synchronizing with the courting male's second flash, the rival male does not mis-time the 2-second courtship flash interval so essential to obtain a female's response. Furthermore, he can induce the female to aim her answer in his direction (Carlson and Copeland, 1983, and unpub.). Frequently the female does answer toward the rival male. The function of the flash sometimes emitted by the rival male after the female's response, the "transvestite flash" of Lloyd (1984), is unknown. Experiments reveal that it is timed from the courting male's second flash and represents a repetition of the courting rhythm. It appears, then, that the rival male has timed incorrectly from the original courting male's second flash instead of from his first flash. This is an example of the kind of perceptual miscue that plagues firefly courtship. Although this competing flash does not normally induce another response from the female, she will often reorient her subsequent answers toward the intruding male (Carlson and Copeland, 1983, and unpub.) The type of flash activity just described portrays "normal" behavior at the peak of the nightly activity period. However, both males and females require a preliminary period of flashing before they are capable of the stereotyped flash periods and flash responses of normal dialogue. For example, we have observed that following a period of flash quiescence, many Photinus macdermottimales emit a series of flash pairs of variable but increasing period before they attain the normal and flashes (Carlson period between Copeland, unpub.). One such male produced a series of paired courtship flashes of increasing interval beginning at intervals of 1625 msec, and finally stabilized at a 2000 msec interval after eleven flash pairs. This same male, when attempting to compete with another courting male during this period, first appeared to interject flashes 1450 and 1600 msec after the courting male's first courtship flash. Eventually, he synchronized [VOLUME 60 his competing flash with the courting male's second flash when his own courtship flashes attained the 2000 msec interval. If exposed to a long quiescent period with no flashing, Photinus macdermottifemales will normally answer any flash, even that of a lighted match or lightning. Additionally, many females when beginning the evening courtship, answer consistently after both the first and second male flashes. In one case, the female answered 5 of the first 15 counterfeit courtship flash pairs after both the first and second flashes, but she answered after both flashes only once in the next 41 courtship pairs (Carlson and Copeland, unpub.). Thus, either the female's ability to discriminate the flash interval or her ability to respond is strongly affected by a previous photic input. Carlson et al. (1977) observed that females routinely responded to the first flash pair of an inappropriate period following a number of pairs of proper period, all of which she had answered. Alternatively, when subjected to a series of inappropriate flash pairs, the female failed to respond to the first pair of the proper period. This observation suggests, then, that both the firefly's state of arousal and past history are important in assessing flash behavior. From computer analysis of the responses of Photinusmacdermottifemales to a wide range of counterfeit flash patterns, Soucek and Carlson (1975) developed a model to explain female flash-response behavior. The model involves an alternating series of excitatory and inhibitory periods initiated by the incoming male flash. This response function, combined with a short-term memory of previous flashes and background noise, determines the probability of particular female responses. The actual responses of females to counterfeit flash sequences show good agreement with those predicted by the model (Carlson and Soucek, 1975). The courtship protocol of P. macdermotti fireflies is one of the most complex described for a Photinus species. It is not clear whether the complexity is unique to this species or simply reflects the greater attention investigators have paid to P. macdermotti.The behavioral nuances that this species exhibits, such as (1) males changing the flash interval between patrolling and courting, (2) the abil- DECEMBER FLASH COMMUNICATION 1985] ity of rival males to synchronize, (3) the changes in flash response by females when presented with different male flash patterns, and particularly (4) the effect of previous flash history on flash behavior, are likely to have counterparts in some or all other Photinus species. With this in mind, it is essential that the courtship flash protocols of firefly species be examined under carefully controlled conditions so that the firefly's entire courtship repertoire is well understood. Only then is it possible to assess the functions of the various flash behaviors observed. ROLE IN OF FIREFLY THE NERVOUS FLASH SYSTEM COMMUNICATION Our description of the physiological processes that underlie the receiving and sending of flashes by fireflies is not meant to be exhaustive. An understanding of the physiology is necessary, however, to allow an assessment of the capabilities, limitations, and variations of firefly flash behavior. For a detailed historical review of research on firefly flash control the reader is directed to Buck (1948), and for more recent information to Case and Strause (1978). Anatomical Organizationof the Adult Lantern and Its Innervation The ultrastructure of the male Photurislantern has been described by Beams and Anderson (1955), Kluss (1958), and Smith (1963). It is organized into a ventral photogenic layer and a dorsal reflector layer. Uniformly spaced tracheae and nerves penetrate ventrally into the photogenic layer and form vertical "cylinders" around which are arranged rosettes of photocytes. Tracheal processes, enclosed in tracheolar cells, project horizontally away from the tracheal cylinders, pass through mitochondria-filled tracheal end cells, and run between the photocytes. Axons leave their surrounding sheath cells and terminate in pad-like endings between the tracheal end cells and the tracheolar cells (Smith, 1963). The nerve endings contain both small electron-clear vesicles and large, electron-dense vesicles. Synaptic specializations appear between the nerve endings and the tracheolar cells, and the latter cells therefore appear to be the elements that couple neural activity to photo- 421 cyte response and may be capable of generating propagated potentials themselves (Case and Strause, 1978). The photocytes are highly structured. They have a clear, outer, differentiated zone and an interior filled with photogenic granules believed to have peroxidase activity (Hanna, Hopkins, and Buck, 1976). These granules contain internal tubular processes (Smith, 1963) and the light-emitting molecule, luciferin (Hopkins and Hanna, 1972; Smalley, Tarwater, and Davidson, 1980), and luciferase (Neuwirth, 1981). By the use of image-intensified cinematographics, Hanson, Miller, and Reynolds (1969) have determined that the smallest functional luminescent unit in the lantern is a "microsource" composed of three photocytes. The light-emitting reaction involves the interaction of reduced luciferin (LH2), luciferase (E), Mg++ and ATP to form an activated (LH2-E-AMP) complex and pyrophosphate (McElroy and DeLuca, 1978, 1981). This complex is oxidized by oxygen to form an oxidized complex (L-E-AMP), with the emission of light. In the light-emitting reaction, one quantum of light is emitted for every luciferin molecule oxidized (Seliger and McElroy, 1960). Seven dorsal unpaired median (DUM) neurons, which reside in the sixth and seventh abdominal ganglia, represent the final common motor pathway from the central nervous system (CNS) to the lantern (Christensen and Carlson, 1981). These cells, three in the sixth and four in the seventh abdominal ganglia, have a structure that is characteristic of DUM neurons identified in other insects (Hoyle, Dagan, Moberly, and Colquhoun, 1974). Projecting dorsally from each large soma is a neurite, which bifurcates into two bilateral axons that leave the ganglion and ramify extensively throughout the photogenic tissue (Christensen and Carlson, 1981). This arrangement is capable of synchronizing the excitation of thousands of photocytes to produce the coordinated flash of the male. Motor (Neural and Pharmacological) Controlof the Flash The adult firefly's lantern has the characteristics of a true neuroeffector similar to 422 THE QUARTERLY REVIEW OF BIOLOGY muscle (Chang, 1956; Buck and Case, 1961). It responds to direct electrical stimulation analogously to muscle, and shows facilitation, summation, constant stimulus-response latency, tetanus, treppe, adaptation, and fatigue (Buck and Case, 1961). The topographical organization of the lantern's luminescence closely follows its gross neural organization (Hanson, 1962). Direct electrical stimulation of the photogenic tissue produces two kinds of flash responses depending upon the voltage of the stimulus (Buck, Case, and Hanson, 1963). At lowstimulus voltages, a flash of 70 msec. latency appears; at high voltages there is a "quick" flash of 18 msec. latency. In the former case, the lantern appears to be activated by way of its nerves, but the quick flash is presumably caused by a more direct activation of the photogenic tissue that bypasses the nerves. Initial studies of the pharmacology of the adult lantern implicated an adrenergic transmitter (Smalley, 1965; Carlson, 1968a), but in the larval lantern the monophenolic amines, synephrine and octopamine, were found to be even more potent (Carlson, 1968b). The photomotor neurons, like the DUM neurons of other insects (Hoyle, 1975; Evans and O'Shea, 1977) are now believed to contain octopamine as a neurotr ansmitter (Robertson and Carlson, 1976; Copeland and Robertson, 1982). This concept has been strengthened by the demonstration that octopamine is not only found in the larval lantern, but also in the lantern ganglion (8th abdominal) and in the photomotor neurons themselves (Christensen, Sherman, McCaman, and Carlson, 1983). An octopaminesensitive adenylate cyclase has been found in the larval lantern (Oertel and Case, 1976). Exogenously applied octopamine stimulates the production of enormous quantities of cyclic AMP (Nathanson, 1979); Nathanson and Hunnicutt, 1979), which is believed to act as a second messenger in the induction of luminescence. The remaining steps in the chain of events from neural input to luminescence are unknown. Complicating the search is the fact that any manipulation of the adult lantern that admits oxygen induces an uncontrollable glow. Oxygen control, therefore, appears to play a major role in the ability of the adult lantern [VOLUME 60 to make a rapid, brilliant burst of luminescence. Favoring this interpretation, Ghiradella (1977, 1978, 1983) had reported that the lantern tracheoles contain unusual strengthening structures that may insure an access of oxygen to the photogenic tissue by resisting collapse when fluid is absorbed from the tracheoles. DeLuca and McElroy (1974) reported that mixtures of luciferase and substrate, allowed to form the active complex, reach peak luminescence in only 60 msec. following injection of oxygen. Several hundred milliseconds are needed if the reaction is initiated by combining luciferin with luciferase and cofactors in the presence of oxygen. Although we do not understand the final coupling mechanisms, the knowledge that the brain, operating via the photomotor neurons, can precisely control the flash of the firefly, is sufficient to identify flash behavior as a true reflection of the output of timing circuits (pattern generators) residing inrthe firefly brain. SensoryReceptionof the Flash The firefly's flash is a signal that must be clearly distinguished from visual interference by ambient light. Flashing activity, therefore, is restricted to periods of low illumination. For example, Dreisig (1975) has shown that the peak of flashing activity by fireflies in different geographic areas does not begin before the environmental illumination levels have dropped below 1 lux. The firefly's compound eye is apparently also tuned to the emission spectrum of its own species' flash. The luminescence spectra of North American fireflies have maxima in the yellow-green spectral band (Seliger, Buck, Fastie, and McElroy, 1964; Biggley, Lloyd, and Seliger, 1967). By means of the electroretinogram two sensitivity peaks were found in the eyes of Photinus pyralis (Lall, Chapman, Trouth, and Holloway, 1980) and Photinus macdermotti(Lall, pers. commun.). One peak is in the near ultraviolet, the other in the longer wavelengths close to the emission peak (565 nm) of the flash of those species. The longer-wavelength sensitivity peak in the eye of Photuris versicoloralso corresponds to the emission peak of its green (552 nm) flash (Lall, 1981). Lall, Seliger, Biggley, and Lloyd (1980) demonstrated a DECEMBER 1985] FLASH COMMUNICATION correlation between the firefly's emission spectrum and the light level during courtship activity. Yellow-flashing fireflies tended to be active only at dusk while green-flashing species did not become fully active until full darkness. Seliger, Lall, Lloyd, and Biggley (1982a, b) pointed out that at dusk there is a large amount of reflected green light from vegetation and that consequently flashes in or approaching the yellow region of the spectrum will stand out in contrast to this reflected green color. The firefly eye itself contains further specializations that increase its sensitivity. The ommatidia are constructed to sum light from several facets while also preserving some directional information from the source (Horridge, 1969). Not only are the eyes and facets of luminescent lampyrids generally larger than those of non-luminescent species, but the eyes and facets of dark-active fireflies are larger than those of dusk or active forms (Case, 1984). The brain of the firefly shows specializations for processing luminescent signals: in Phausis splendidulathe optic lobe of the brain reflects the large size of the eyes (Ohly, 1975). The incoming flash that is transduced by the eye can be complex in form and timing (see Fig. 1), and some of the timing parameters may be important in flash codes. However, such features of the flash as its color, intensity, or movement have been shown to be unimportant (Lloyd, 1966a). In Photinus fireflies, Lloyd (1966a) conclusively demonstrated that timing parameters (the male's flash interval and duration and the female's flash duration and response latency) play important roles in species recognition and sexual recognition. The neural correlates of most of these stimulus features have not yet been studied. Many male courtship flashes contain characteristic intensity modulations (see Figure 1). Lloyd (1973b) had pointed out that although absolute flash intensity is of little use, since it varies with distance, the fixed relative intensities of bimodal courtship flashes, for example in Southeast Asian fireflies, would be preserved over distance and therefore serve as excellent parameters for flash recognition. As other examples, the Luciola lusitanica female triggers her responses from the on- 423 set of the rapidly brightening male flash (Papi, 1969), and Photuris lucicrescensfemales trigger their responses from the slowly rising intensity of the male crescendo flash (Carlson, Copeland, and Shaskan, 1982). Unmated P versicolorfemales require at least two interpulse intervals of less than 300 msec before responding to a male's courtship flash. ANALYSIS OF OF THE THE CENTRAL FLASH PROCESSING INPUT Studies made of the courtship flash patterns of North American lampyrids by McDermott (1910-1917), Buck (1937), Green (1956), Buck and Buck (1972), Lloyd (1966a), and others, of a European firefly, Luciola lusitanica by Papi (1969), and of Japanese fireflies by Ohba (1983), among others, have revealed the stereotyped nature of photic communication systems. Barber (1951), in his study of Photuris fireflies in the Baltimore area, emphasized this fact by means of a concept that he termed a "physiological species." This concept recognizes that the dialogue flash pattern is as useful a taxonomic tool as are morphological characters. However, the complexities of the Photuris flash code make the use of flash behavior as a taxonomic tool difficult (Lloyd, 1969a). The studies of Buck, Case and Hanson, reviewed by Carlson (1969), have revealed that the firefly flash provides a window into the insect's brain activity. Case and Buck (1963) were able to monitor the neural input to the lantern by means of electrodes inserted into the lantern. They found that the overall pattern of luminescence intensity corresponded rather well to the structure of the incoming neural burst. The correspondence of the burst with the flash is not exact, however, because the recording electrodes in the lantern sample only a small portion of the neural input, whereas the phototube records the luminescence produced by the entire lantern (see Fig. 1). Case and Buck (1963) also found that the overall luminescent activity of an individual closely mirrored the firefly's excitatory state. Refractory animals could be made to flash only with difficulty. Initially, the lantern glowed and scintillated with unstructured luminescence. Animals in this state were characterized by very high electrical thresholds, 424 THE QUARTERLY REVIEW OF BIOLOGY dim induced luminescence, a lack of spontaneous flashing and lethargic behavior. Responsiveness could be restored by handling the firefly or by other irritation. Arousal had to precede decapitation, however, a fact suggesting that the level of brain excitability determines the responsiveness of the entire excitation pathway. Case and Buck (1963) speculated that prior to flashing, the Photuris lantern requires neural priming before wellshaped courtship flashes can be produced. Once both male and female Photuris fireflies become aroused, they could flash spontaneously for hours. Excited Photuris versicolormales often emit simple, rhythmic flashes that are unlike their rapidly modulated courtship flashes. These simple flashes do not elicit responses from conspecific females. P versicolorfemales are often observed flashing spontaneously as they walk through the underbrush, and these females do not respond to flash signals of any kind (Barber, 1951). Luminescence, therefore, is to be observed in non-courtship situations during which the animal is agitated or is actively moving. Although males of most American Photinus fireflies produce flashes of a relatively simple time course, the courtship flashes of Photuris males can be intensity-modulated. The crescendo flash of Photuris lucicrescens,a slowly brightening glow culminating in a brilliant burst of light, is shaped by a long, continuous neural burst, probably of increasing spike frequency (Carlson, Copeland, and Shaskan, 1982). The courtship flash of P versicolor (Carlson, 1981) is composed of three pulses, usually of descending intensity, and is shaped by three neural bursts of descending spike number (see Fig. 1). As is illustrated in Fig. 1, the flash-generating center in the brain of P versicolormale fireflies is capable of producing a stereotyped pattern of neural bursts in which spike number, spike frequency, and burst period are relatively constant. Fluctuations in these neural parameters do occur, however, as is also shown in Fig. 1. Not only do individual bursts vary in spike number, but single courtship flashes also can vary from two to many pulses. The consequence of these inherent fluctuations for the effectivness of communication can only be determined by an analysis of courtships under controlled conditions. [VOLUME 60 In the absence of the central nervous system, the lanterns of some fireflies of the genus Photurisrespond differently to direct electrical stimulation. In deganglionated lanterns of P lucicrescensmales, it is possible to alter the time course of luminescence by changing the frequency of electrical stimulation (Carlson, Copeland, and Shaskan, 1982). Deganglionated P versicolorlanterns respond only with short flash pulses of approximately 100 msec duration, no matter what stimulus parameters are used. For example, a 500-msec stimulus train composed of 1-msec stimuli of 100 Hz frequency results in a triple-pulsed flash of descending pulse intensity. This flash differs from the courtship flash only in having shorter interpulse intervals. Stimulation for longer periods simply produces paroxysms of flash pulses, but no glowing (Carlson, 1981). The lanterns of these two species also differ in their response to solutions in which sodium is replaced by potassium: P versicolor lanterns scintillate vigorously in such a solution (Carlson, 1967) whereas those of P lIuczcrescensrespond only with a glow (Carlson, unpub.). In intact Luciola lusitanica males, Buonamici and Magni (1967) observed two responses to direct electrical stimulation with single, 10msec stimuli. A flash of 100 to 160 msec latency that was kinetically identical to the spontaneous flash was followed by a flash of variable latency (220-750 msec) and variable contour. The latter response could not be induced in decapitated males, however, and it was attributed to reflex activity elicited by stimulation of some afferent pathway. Magni (1967) reported that spontaneous flashing could be depressed by photic stimulation of high intensity and could be facilitated by low levels of illumination. Flashes evoked by direct stimulation of the lantern could also be inhibited by strong photic stimulation, and this effect was mimicked in decapitated males by low-frequency stimulation of the ventral nerve cord. Brunelli, Buonamici, and Magni (1968a) demonstrated that illumination of the eyes of one male could depress the intensity and rate of flashing of a second firefly when their body cavities were connected by a saline bridge. Ablation of the gonads eliminated this inhibitory effect induced by illumination and by low-frequency ventral nerve cord stimulation DECEMBER 1985] FLASH COMMUNICATION (Brunelli, Buonamici, and Magni, 1968b). Perfusion of the body cavities of fireflies with a homogenate of gonads reestablished the inhibition (Brunelli, Buonamici, Magni, and Viola-Magni, 1970). The inhibitory substance was located in granules derived from the cortex of the male gonad. Cortical granule number was reduced in illuminated males, and isolated granules produced the same inhibitory effect as whole-gonad extracts. Noradrenaline, which is present in large amounts in the gonadal granules, induced flash inhibition and was postulated to be the active, inhibitory substance (Bagnoli, Brunelli, Magni, and Viola, 1972). By using restrained preparations, some properties of the central processing system have been revealed via electrical or photic stimuli delivered to the firefly's eye. Electrical stimulation of the eye of a Photurismale with simulated photic input could inhibit or enhance flashing, depending upon the relative intensities of eye and brain stimulation (Case and Buck, 1963). In Photurisfemales this photic inhibition caused by photic stimulation peaked at about 300 msec, then changed into a period of increased excitation manifested as a lowered threshold to brain stimulation (Case and Trinkle, 1968; Carlson and Copeland, 1972). In Luciola lusitanica photic stimulation of the eyes of both males and females induced not inhibition, but a flash response of 250 msec latency (Papi, 1969; Brunelli, Magni, and Pellegrino, 1977), and direct electrical stimulation of the eye mimicked this response in females (Papi, 1969). Brunelli, Magni, and Pellegrino (1977) found that weak photic stimuli induced a later response of 600 msec latency in males, and they concluded that each photic stimulus exerts a dual action on the photomotor centers of the protocerebrum. They suggested that the neural mechanisms underlying reflex flashing are identical in both sexes. In one of the most extensive studies of central control, Bagnoli, Brunelli, Magni, and Musumeci (1976) investigated the neural mechanisms underlying flashing in Luciola lusitanica by using localized electrolytic lesions and electrical stimulations. Electrical stimulation of the median protocerebral neuropil induced fully formed flashes, whereas lesions to this area abolished spontaneous flashing. Separation of the protocerebrum from the 425 optic lobes stopped rhythmic flashing, which was replaced by a continuous dull luminescence. Concomitantly, the flash-inducing photic nerve volleys in the lantern nerves were replaced by an asynchronous, lowfrequency discharge of these nerves. Ablation of the retina-lamina ganglionaris complex resulted in a marked increase of flash frequency, and stimulation of the more central optic ganglion, the medulla, facilitated flashing. From these and other findings these investigators concluded that the rhythmic activity of the central photic neurons, located in the deep protocerebral neuropil of the firefly's brain, results from an oscillator located in the optic lobes. The retina-lamina complex exerts a tonic inhibitory influence on flashing, whereas the medulla induces a phasic facilitatory influence. To explain the response patterns of predatory Photuris versicolorfemales, Soucek and Carlson (unpub.) have developed a quantitative model based upon generation of a sequence of time windows. Using a similar approach to their earlier model for Photinus macdermotti(Soucek and Carlson, 1975), this model can predict the female's response patterns with considerable precision. The model proposes that the response patterns of these females are the result of the entrainment of brain oscillatory circuits that have fixed periods, but can be influenced by the female's previous flash history. Fireflies, then, probably have brain circuits that produce flashes patterned in the domain of time. The synchronously flashing fireflies of Southeast Asia represent an excellent example of the e-volution of precision in such firefly timing circuits. They have provided ideal material for the analysis of flash control by the nervous system (Buck, Buck, Hanson, Mets, and Atta, 1981). The analysis of flash synchrony in fireflies will be described next. The Analysis of CentralProcessingin SynchronouslyFlashing Fireflies Nothing emphasizes the importance of timing patterns and oscillators in firefly flash codes as well as synchronous flashing does. While both Photinuspyralis and Photinus macdermottimales show some propensity for synchronous flashing during courtship, Southeast Asian fireflies of the genera Pteroptyxand Luciola have carried this behavior to a spec- 426 THE QUARTERLY REVIEW OF BIOLOGY tacular extreme, when entire trees containing a swarm of males pulsate rhythmically with light. As described by Buck and Buck (1966, 1968, 1976, and 1978), males congregate in large numbers in trees along river banks and flash at frequencies of 1 to 4 hz, depending on the species, but in total synchrony. In Malaysia and Thailand, synchronizing males of Pteroptyxmalaccaeappear to be evenly spaced within the mangrove trees and exhibit a flash period of 500 to 600 msec. Photomultiplier recordings of their double-pulsed flashes show such complete coincidence that the shape of the individual male's flash is easily discerned within the massed display. In another Malaysian species, Pteroptyx tener, which synchronizes at about 3.7 Hz (Case, 1980), the males may recognize females by means of the latter's nonsynchronized flashes, which should stand out from the background display. Prior to attempting copulation, the male mounts the female, twists its lantern over her head, and flashes in synchrony with other males, directly into her eyes. In his comparative studies of such firefly pacemakers, Hanson (1978) has suggested that at least two different mechanisms exist to attain synchrony. It could be attained either by a cycle-by-cycle phase shift of a stable oscillator or by a change of the underlying period of a variable oscillator. The synchronously flashing New Guinea firefly, Pteroptyxcribellata, is an example of a species using the first mechanism (Hanson, Case, Buck, and Buck, 1971; Buck, Buck, Case, and Hanson, 1981). It emits free-run flashes at nearly 1 Hz. A flash from another male seen within a critical period after flashing resets the first male's next flash cycle to the beginning. If the two males' flash periods are very similar, the reset flash by the follower male coincides with the next flash fromi the pacemaker male, and synchrony results (Hanson, Case, and Barnes, 1972; Hanson, 1978). P malaccaeand P tener use a different mechanism to achieve synchrony. They can synchronize with an out-ofphase pacer flash by slightly shortening or lengthening their flashing periods so that coincidence develops over the course of several cycles (Hanson, 1978). These males are continually resetting their flashes to achieve more precise synchrony. This ten- [VOLUME 60 dency to synchronize is so strong that males will continue to flash in synchrony with their conspecifics even during flight or while mating. Photinuspyralis males can synchronize their courtship flashes emitted every six seconds, particularly if they are densely aggregated (Buck, 1935; Buck, Hanson, Buck, and Case, 1982). In this case, however, the resetting male pacemaker flash triggers a flash from the synchronizing male within less than 400 msec. Since the P pyralis flash normally lasts 600 msec, both flashes overlap and appear to synchronize. This is a different mechanism from that of New Guinea Pteroptyxmales, in which the pacemaker flash resets the nextflash period of the follower male. Sychrony is probably necessary to preserve the timing patterns essential for sexual and species recognition in an environment crowded with many individuals (Otte, 1980). Although males and females can direct their flashes by twisting their lanterns toward flash sources and therefore have some directional sense, they apparently cannot discriminate between spatially separate light sources that occur simultaneously. Therefore, flashes entering one eye can easily destroy the timing pattern of flashes entering the other eye. Case (1984) has shown that in Pteroptyxtenermales synchrony can be established to two spatially separated light generators 1800 out of phase at half the required frequency, summing to make the required frequency. An analogous situation occurs in Photinus greeni females (courtship code similar to Photinus macdermotti) (Case, 1984). The interval between the P greenimale's courtship flash pair is about 1.2 seconds, and the female answers 600 to 800 msec after the second flash. If the female receives two properly timed flashes from one source in one eye and receives a flash from a different source in the other eye during the period between those flashes, she does not respond to either. If she receives two properly timed flashes from two different sources, one in each eye, she responds toward the flash that arrived second. The female's response can be manipulated even after she has received a correctly timed flash pair. A third flash timed to arrive in one eye slightly after two properly timed flashes delivered to the other eye causes the female to respond in the direction of the third flash and 1985] DECEMBER 8 2 _ 6 _ 16 FLASH COMMUNICATION 24 32 40 48 10 _ ir 14 - m 18 _ z - 22 - 26 _ I 34 _ _ 30 _ 38 - 1 42 - w 46 - o 50 cn _ at the proper latency following that flash, that is, at a slightly longer latency than might be expected to follow the normal pair (Case, 1984). Because the eye of the firefly is designed to collect light over a wide area and because of the animal's inability to attend and respond to localized points of light to the exclusion of other light sources, flash communication in dense aggregations is very difficult. In this situation, flash synchrony becomes an extremely useful tool for extracting the timing relationships of male and female flashes. Analysis of CentralProcessing. Flash Entrainment - 54 - 58 - 62 -_ 66 - Additional insight into the operation of central processing is obtained by an examination of a mating-induced change in flash behavior seen in Photuris versicolorfemales (Nelson, Carlson, and Copeland, 1975). Prior to mating, these females respond only to a con16 8 24 32 (FFA) LATENCY To OF RESPONSES 48 OF Afemmefatale PAIRS OF COUNTERFEIT VARYING 40 x 100) TIME (milliseconds FIG. 2. 427 FLASHES A OF INTERVALS The first counterfeit flash is placed at 0 on the abscissa and the second counterfeit flash at the beginning of each horizontal line. Flash pairs were separated by intervals of at least 10 seconds. The first ten counterfeit flashes of 300 msec period induced female responses averaging 2900 msec latency, measured from the second counterfeit flash. Most counterfeit flash pairs induced female responses of 800 to 1100 msec latency (measured from the second counterfeit flash). Response latency to counterfeit flash pairs of greater than 1500 msec was significantly longer. Flash pairs 62 to 64 show a typical response of afemmefatale to abrupt changes in flash parameters. The period of flash pair 63 was longer than 1500 msec, but the femmefatale exhibited a short latency (1040 msec) response, characteristic of answers to flash periods of less than 1500 msec. The counterfeit flash period and female flash latency were measured from the onset of each flash. The females included in these tests and in Fig. 3 and Tables 2 and 3 were each maintained in a terrarium in the field. Counterfeit flashes were produced by a stimulator-driven flashlight and female responses were detected by a photomultiplier, the output of which was fed to an FM tape recorder. Tape recorder records were transferred to a Beckman Dynagraph pen recorder (Fig. 3), and the measurements were made from the records. Temperature, 21?C. D Is FIG. 3. EXAMPLES OF ENTRAINMENT OFfemme fatale C's (FFC) (SEE TABLE 3) RESPONSES TO MULTIPLE COUNTERFEIT FLASHES The female'sresponses are marked with circles. Counterfeit flash period in msec is as follows: (A) 500, 520, 312. (B) 1280, 1240, 1250, 1332, 1400. (C) 420, female latency 640 msec to 3rd, 4th, and 5th counterfeit flashes. (D) 560, female latency 750 msec to 2nd, 3rd, 4th, and 5th counterfeit flashes. Note the characteristicintensity pattern of female responses. Time scale: 1 second. Temperature, 22?C. Counterfeit flashes of 150 msec duration have been cut at the top by the recorder. Experimental protocol, as described for Fig. 2. THE QUARTERLY REVIEW OF BIOLOGY 428 TABLE 1 Responselatency(RL) of femme fatale B (FFB) to stimulusflashes (SF) SF Interval SF RL after Last SF (msec) (msec) number (x) (S. D.) (n) 420 440 516 1100 1600 2168 1400* 2 3-5 2 2 2 2 1 3022 715 836 770 899 2286 1027 37.3 51.8 12.9 46.2 45.0 190.8 68.3 7 45 8 29 5 11 12 * Rhythmically repeated flashes. Interval between paired and multiple flash sets was over 5 seconds. Paired flashes as illustrated in Fig. 3A, rhythmicallyrepeated flashes as in Fig. 3B, and multiple flashes as in Figs. 3C and D. See Fig. 2 for description of experimental protocol. Temperature, 21?C. specific male's triple-pulsed courtship flash, by answering approximately 1 second after the end of the flash. In this unmated state, the stationary female answers male courtship flashes from a hunched posture, while twisting her lantern toward the male flash. Within three days after mating, the female responds very infrequently to a conspecific male's courtship flash, but she does respond to a wide range of other flash patterns. In this behavioral mode the female adopts an upright, prey-capture posture and is highly mobile. With appropriate flash responses these voracious insects are capable of attracting heterospecific males, then capturing and devouring them. In this predatory state, mated females that respond to flashes of heterospecific males have been called femmesfatales (Lloyd, 1965) and their behavior has been proposed as an example of aggressive mimicry (Wickler, 1968). The attractiveness of Photuris females in these two mated states is illustrated by observations of a femmefatale and an unmated female placed in adjacent terraria in the field. At one time, nine conspecific males were observed on the unmated female's terrarium, whereas the femmefatale did not attract a single male all evening (Carlson and Copeland, unpub.). Barber (1951) observed Photuris females walking through the underbrush and flashing spontaneously, apparently oblivious to exog- [VOLUME 60 enous flashes. He suggested that these females had already mated. We have found that such females, oblivious to flashes at first, become femmesfatales when tested again one to three days after capture. Zorn and Carlson (1978) observed that unmated females, mated to sterile males, remain in the courtship posture but no longer respond at all to flashes. These unfertilized females did not become femmesfatales. Zorn and Carlson (1978) speculated that mating causes the females to stop answering conspecific male courtship flashes, but something transferred by the male, such as sperm, is required to induce predatory mimic behavior. It appears that the flash-control circuits of femmesfatales can be triggered by a wide array of flash patterns. Femmesfatalesof Photuris versicolor tested in the field or in the laboratory show highly stereotyped and precise response patterns (Figs. 2 and 3, Tables 1 and 2). They respond to rhythmically repeated flashes (flash trains) with a fixed latency to each of the flashes. Within certain limits, femmefatale flash latency can clearly be triggered by paired flashes at certain interflash intervals. The response behaviors offemmesfatales A (see Fig. 2), B (see Table 1), and C (see Table 2 and Fig. 3) were similar to that shown by 12 other females tested. The responses to stimulus flashes of different patterns and periods can be summarized as follows: (1) At the extremes of the sign.al pair interval (short intervals of less than 320 msec and long periods over 1500 msec) female response latency changes abruptly, but consistently. As might be expected, however, the exact break point is affected by temperature. FFA (see Fig. 2) and FFB (see Table 1) were tested near 21?C, whereas FFC (see Fig. 3 and Table 2) was tested between 220 and 23.3?C. This difference may explain why FFC responded to paired flashes of a 320-420 msec period differently than did FFA or FFB. (2) Single flashes more than 5 seconds apart elicited responses of approximately 3 seconds latency (see FFC, Table 2, Part B). (3) The Photuris versicolor triple-pulsed courtship flash has pulse periods ranging from 220 to 270 msec at temperatures of 23? and 20?C respectively (Carlson, 1981), and the unmated female requires at least two periods (three flashes) before she will respond DECEMBER 1985] FLASH COMMUNICATION 429 TABLE 2 Responselatency(RL) of femme fatale C (FFC) to stimulusflashes (SF) A. RESPONSES TO PAIRED FLASHES 741 666 665 756 775 729 802 988 320 420 560 620 900 1100 1620 1620* 27.1 26.5 40.0 12.8 33.3 24.6 5.4 67.2 17 6 14 27 13 10 5 5 FirstSF of flashpair B. SF Interval (msec.) 5000 940 1050 1450 1600 1900 RESPONSES TO SINGLE FLASHES FLASHES (FIG. 3B) Response Latency to Stimulus Flashes (msec.) (x) (S.D.) (n) AND REPEATED Type 2858 786 773 795 879 904 Single Trains t it t it C. SF Period (msec.) 420 520 746 3A) Response Latency after Stimulus Flashes (msec) (x) (S.D.) (n) SF Interval (msec) * (FIG. RESPONSES SF 2 738 746 TO MULTIPLE FLASHES (FIG. 74.1 39.2 61.7 54.0 37.2 40.4 3C AND D) Response Latency to Stimultis Flashes (msec.)* SF 4 SF 5 SF 3 640 706 757 643 735 760 11 17 52 96 6 21 647 SF 6 646 760 * Rangeof standarddeviations10.4-19.0. Restperiodbetweenpresentations of pairedandmultipleflashsetswas5 secondsorlonger.SeeFig. 2 fordescription of experimentalprotocol.Temperature, 22.0?-23.30C. (Zorn and Carlson, 1978). Femmesfatales appeared unable to resolve periods that short (see FFA, Fig. 2; FFC, Table 2). They respond to double and triple-pulsed flashes of less than 420 msec interflash interval in identical fashion to single flashes, with response latencies of about 3 seconds measured from the onset of the second stimulus flash. It is possible thatfemmesfatales cannot resolve flash pairs 400 msec or less apart and therefore respond as if they were observing single flashes. (4) Paired flashes 500 to 1400 msec apart induced responses of 650 to 900 msec latency (see FFA. Fig. 2; and FFB and FFC, Tables 1 and 2). Flash trains with periods in the same range induced responses in the 650-1000 msec latency range (see FFB, Table 1; FFC, Table 2 and Fig 3). In this timing range the latency of responses remained relatively constant with increasing period. (5) With stimulus flashes of 1500 msec or more apart, individual females abruptly and consistenly changed their response patterns and latencies. FAA (see Fig. 2) abruptly increased her latency from 700 to 2700 msec to the second flash of pairs more than 1500 msec apart. FCC (see Table 2) responded after both flashes of a pair 1620 msec apart. FFB (see Table 1) responded to paired stimulus flashes 2168 msec apart with a latency to the second of over 2 seconds. The courtship flash pairs of Photinus macdermotti males are 430 THE QUARTERLY REVIEW OF BIOLOGY reported as being approximately every 2150 msec at 21?C. Having a female response latency of over 2 seconds, FFC would attract but few P macdermottimales, for the conspecific female answers with a latency of 1.5 seconds. (6) Femmesfatales are entrained to successive flashes at short intervals (400-800 msec) with latencies of considerable precision, as shown by the small S.D.s of FFC, Table 2, part C. (7) As illustrated in Fig. 2, the response behavior of Photurisversicolorfemales is strongly affected by the animal's previous flash history, an effect previously observed in Photinus macdermottifemales (Carlson et al., 1977). FFA usually responded to flashes more than 1500 msec apart with response latencies of over 2 seconds. At flash pair 63, an abrupt interval change occurred from 1360 msec to over 1600 msec, but FFA responded to the longer interval with a latency of only 1 second. Summary. PhysiologicalProcesses(Motor, Sensory, Central)that ControlFirefly Flashing The picture that emerges from the physiological observations on the control of flash behavior is as follows: (1) The courtship flash behaviors of fireflies are controled by neural pattern generators that produce stereotyped flash communication patterns of minimum ambiguity. These pattern generators have been perpetuated by selection and result in unique flash codes that contribute to the reproductive isolation of each species. In some species, these pattern generators can be activated directly by electrical stimulation of the firefly's brain (Bagnoli et al., 1976, in Luciola lusitanica;Case and Trinkle, 1968, and Carlson and Copeland, 1972, in Photuris). (2) Fireflies may be programmed to produce only one or a very few flash patterns. Photinusmacdermottimales are capable of emitting two different flash patterns during different stages of courtship, either rhythmic flashes during searching or paired flashes during courtship (Lloyd, 1969b; Carlson et al., 1976). (3) Neural priming of flash-pattern generators must occur for the system to operate with precision. Male fireflies must undergo a [VOLUME 60 period of neural warmup before properly shaped flashes (Case and Buck, 1963) and properly timed flashes (see Table 1) can be produced. Even in suitably primed males, random fluctuations at each step in the chain of flash-excitation processes may degrade the timing of the signal (see Fig. 1). Furthermore, female fireflies must be primed to respond to the correct courtship pattern with the appropriate latency. The level of priming can fluctuate so that behavior is strongly influenced by the immediately previous flash history (Carlson et al., 1977, in Photinus macdermotti females). This effect is most apparent when females are subjected to sudden changes of flash pattern (Carlson and Soucek, 1975, for Photinusmacdermotti;see Fig. 2, for Photurisversicolor). (4) The ability of fireflies to flash is greatly influenced by their state of arousal. At low levels of arousal (flash quiescence during daytime), it is difficult to induce a normal courtship flash (Case and Buck, 1963). We have observed in addition that highly excited fireflies produce paroxysms of flashes that have no apparent communicative significance. In Photuris fireflies, the flash-generating center appears to be easily activated by noncourtship interference caused by movement of the animal (Barber, 1951, reported that females flash spontaneously as they are observed walking through the underbrush) or by undergoing physical manipulation (fireflies flash vigorously when trapped in spider webs or when handled or restrained, Case and Buck, 1963). (5) The firefly's visual apparatus is adapted to receive and to process flash signals (Lall, 1981; Horridge, 1969). The eyes collect light through wide visual angles, and the spectral sensitivity of the firefly's eye closely approximates the emission spectrum of its flash. (6) The neural pattern generators of some male and of all female fireflies can be activated by light flashes, so as to result in female responses of proper latency (Soucek and Carlson, 1975, in Photinusmacdermotti;Soucek and Carlson, unpub., in Photuris versicolor) and in flash synchrony in rival males (Buck, Buck, Hanson, Mets, and Atta, 1981, in Pteroptyx;Buck et al., 1982, in Photinuspyralis; Carlson and Copeland, 1983, in Photinusmacdermottz).Femmesfatalesof Photurisversicolorre- DECEMBER 1985] FLASH COMMUNICATION spond consistently to a wide array of flash patterns (see Figs. 2 and 3, Tables 1 and 2). Their response patterns can be interpreted on the basis of a series of neural time windows that are susceptible to the memory of previous flashes (Soucek and Carlson, unpub.). 431 criteria. The courtship flashes of Photinus pyralis and Photinus macdermottimales differ significantly from the triple-pulsed flash of Photuris versicolor males. The responses of femmes fatales to the flashes of heterospecific males appear more frequently and consistently than purely random flashes (Nelson, MIMICRY IN FIREFLIES Carlson, and Copeland, 1975). However, the precision of the response latency of femmes The conversion of females from unmated to mated (femmefatale)behavior (luminescent, fatales is questionable (Carlson and Copepostural, and locomotory) by means of an ir- land, unpub.). As illustrated by FFC (see Tareversible mating-induced switch (Nelson, ble 2), single flashes repeated less often than once in 5 seconds can induce response latenCarlson, and Copeland, 1975) has been described previously. The mating of females of cies of about 3 seconds. In the field, however, some Photuris species apparently renders femmesfatales appear to answer at this latency them no longer responsive to courtship, con(3 sec) less often, and usually respond with a trary to the effect in Photinusfemales, who will latency of approximately 1 sec to P pyralis mate and court on successive evenings (Carlmales (J. Buck, pers. commun.). Responses son and Copeland, unpub.). Further, Photuris to Photinus macdermotticourtship flashes are versicolorfemales in the field are eitherfemmes sometimes emitted correctly after the second fatales or are simply unresponsive to flashes of courtship flash, but at 20?C such responses any kind; virtually none are unmated (Zorn are at highly variable latencies rather than at and Carlson, 1978). Only by raising females the 1500 msec characteristic of P macdermotti from larvae in the laboratory (McLean, females. As shown in Fig. 2, the latency of reBuck, and Hanson, 1972) could unmated fesponse of FFA to flash intervals longer than males be obtained that would participate in 1500 msec varied between 2.0 to 2.8 seconds. courtship with a conspecific male (P ver- FFB (see Table 1) responded with latencies sicolor,Nelson, Carlson, and Copeland, 1975; longer than 2 seconds to paired flashes that P lucicrescens, Carlson, Copeland, and mimic the courtship flash period of P macderShaskan, 1982). These laboratory-raised femotti males. In the only direct assessment of males were tested in both the field and the the efficiency of Photuris predation, Lloyd laboratory. (1979) reported that of 119 Photinus collustrans What then is female aggressive mimicry in males he followed, none closely approached fireflies? We feel that to be considered mimor were captured by the many femmesfatales, icry, the flash responses of Photuris females but two conspecific females were quickly apshould fulfill at least three criteria. (1) They proached and mated. A strong case for aggressive mimicry has should be produced in response to male flashes significantly different from those of been made for only three species of Photuris their own males. This criterion would exfireflies. They are P versicolor(Lloyd, 1965; clude the apparent mimicry produced by the Nelson, Carlson, and Copeland, 1975), P jamaicensis (Farnworth, 1973), and P fairserendipitous overlap of conspecific and hetchildi (Buschman, 1974). The flashes of erospecific (mimic) courtship codes. While P pennsylvanica and P lucicrescens males, the latter might represent a form of mimicry, whose females are not known to be aggressive it is not what is usually meant when the conmimics, are of long duration and simple in cept of aggressive mimicry is discussed shape, whereas the males of the known ag(Wickler, 1968). (2) The responses should be produced often enough to be more-than rangressive mimics emit flickering or pulsed flashes. The P versicolor, P jamaicensis and dom, spontaneous flashes of unknown comP fairchildi females must, therefore, be able to municative significance. (3) The responses should be timed so precisely that they can at- analyze rapid light fluctuations. Perhaps, aftract the heterospecific male. ter mating, a change within the brain renders To date, the flash responses of Photuris ver- these fireflies incapable of responding to consicolor femmes fatales fulfill the first two specific males, but still able to entrain to the THE QUARTERLY REVIEW OF BIOLOGY 432 simpler flash patterns of heterospecific males. This, of course, is pure speculation, but it does point up the importance of rigorously establishing the response parameters of any suspected or known aggressive mimic. Until this is done, the evolutinary implications of aggressive mimicry for firefly flash codes must remain uncertain. SUMMARY AND CONCLUSIONS In summary, firefly communication depends on signal systems coded for timing patterns of flashes. These timing patterns are produced by insects with relatively simple sensory and neural equipment that must be primed in order to function with sufficient precision. For lack of a better term, we may say that the firefly's state of arousal must be sufficiently elevated to enable it to produce the proper flash patterns. It is difficult to underestimate the importance of this concept of state of arousal because it is seen in nearly every aspect of firefly flash behavior. The lanterns of both quiescent Photuris and Photinus males require considerable neural input before their courtship flashes can be correctly produced. Much neural priming is required before Photuris males can emit their characteristic intensitymodulated courtship flashes and Photinus males can achieve the correct timing patterns of their courtship flashes. Photinus females may be unresponsive or may respond incorrectly during the initial stages of courtship, but when they become sufficiently aroused they may become highly discriminating. The central flash triggering circuits of the firefly's brain appear to be highly susceptible to activation during periods of high excitability, and the result is often the production of flashes of unknown communicative function. Photuris females, presumably mated, are often seen flashing spontaneously as they walk through the foliage (Barber, 1951). Fireflies that are handled, that crash in flight, or that fly into spider webs flash vigorously. The timing circuits (pattern generators and pacemakers) of the firefly's brain have been studied in a wide array of species. By means of electrical or photic stimulation, firefly brain circuits have been activated to produce the flash patterns observed in the field (Luciola lusitanica males, Bagnoli et al., 1976; [VOLUME 60 Photurisfemales, Case and Trinkle, 1968, and Carlson and Copeland, 1972). By analyzing the responses of fireflies to natural or artificial flash patterns, models have been developed that predict firefly flash behavior with considerable accuracy (synchronously flashing Pteroptyxmales -Buck, Buck, Case, and Hanson, 1981; and Photinus macdermottifemales-Soucek and Carlson, 1975). Essential to the success of these studies has been rigid experimental control combined with a knowledge of the previous flash history of each animal. All of these considerations point to the difficulty of analyzing the flashes of fireflies in the field in situations where these experimental requirements cannot be met. Given our present understanding of the physiological control of firefly flash behavior, questions may be raised concerning the responses of Photurisfemmesfatalesto the flashes of heterospecific males: do they truly represent mimicry? The responses of these predatory females may simply be too inaccurate to meet the criteria of mimicry. Flashes presumed to be mimicking may actually reflect entrainment of neural oscillatory circuits of femmesfatales to a wide array of different flash patterns, and the flash responses that result may have only a superficial resemblance to the response patterns of other species. The prey-capturing ability of these flashes of femmesfatales, such as it is, may therefore only reflect the wide timing variations in the flash dialogues of the prey species. The choice between two such possible explanations offemme fatale behavior can only be made after rigorous experimental examination in which the response latencies of predatory females to different flash patterns have been measured under conditions in which the previous flash history of each female is known. The firefly's flash communication system, in short, is designed to emit pulsed signals in precise timing patterns that convey courtship information. The variation observed in these patterns is the result of normal fluctuations found in any physiological system. It is possible to misinterpret this variation as being of functional significance if the observations of flash behavior are not performed under controlled conditions. Without a knowledge of the firefly's central excitatory state and previous flash history, it is very difficult to inter- DECEMBER 1985] FLASH COMMUNICATION pret the precise function of the flash behavior observed. We think that the study of flash communication in fireflies is really the study of flash entrainment. Male fireflies entrain to the flashes of others, so as to produce synchronous flashing by means of different mechanisms in Photinus macdermottirivalry, Photinus pyralis rivalry, and Pteroptyx communal flash displays. Photinus macdermottifemales entrain to the rhythmic flashes of Photi- 433 nus macdermottimales. 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