Fireflies & Glow-worms

Lampyrid 2017 4,1-19 | PDF

Analysis of courtship flash behaviour in two Photuris fireflies (Coleoptera: Lampyridae) with field validation and rearing notes

Lawrent L. Buschman

Department of Entomology, Kansas State University, Manhattan, KS USA 60605

Abstract

The fireflies Photuris versicolor quadrifulgens Barber and Photuris tremulans Barber from East Tennessee were reared in the laboratory from eggs through to adults. Duration to pupation, body mass and sex ratios were recorded. Virgin adult females were used for courtship studies recorded using video equipment. Analysis began with discriminating flashes associated with courtship communication from those which are not. A responsive virgin female would respond to some of the male flashes. These dialogues identified the male “advertising flashes” and the female “response flashes”. Other non-dialogue flashes were considered “periodic flashes”. In P. quadrifulgens, the male advertising flash pattern was two to five flashes emitted ca. 0.7-sec intervals. The female response was a flash train with one to nine flashes emitted at ca. 0.2-sec intervals. The female flash train often started during the third male flash. Males seemed to synchronize flashes with each other when one male was in a flash dialogue with a female. The P. tremulans flash dialogue was a continuing duet of male and female flashes. The male flashed once in ca. 3 sec while the female flashed ca. two times in 3 sec. The female response flashes were ca. 1-sec apart and started ca. 1 sec after the male flash and her third flash sometimes synchronized with the second male flash. Male P. tremulans did not seem to synchronize with each other. The “periodic flashes” had a frequency distribution with a peak at ca. 1 sec. They seemed to have some periodicity so they are being called “periodic flashes”, a separate type of flash. Periodic flashes were produced by both males and females as they move about in the jar, cage or on vegetation. The function of periodic flashes is unknown, but they would seem to be perfect aposematic warning signals to keep predators at bay. These are some of the first detailed descriptions of courtship communication in Photuris fireflies.

Key words: courtship flash behaviour, flash-answer, flash duet, growing degree days, Photuris quadrifulgens, Photuris tremulans, synchronized flashing

Introduction

Courtship flash communication in the Photuris fireflies is poorly understood because it is so seldom observed in the field, and when it is observed, it happens so fast that the observer is left wondering what happened (Buschman 1974, Buschman 2017). Only a few observations of Photuris courtship communication have been analysed in any detail: Buschman (1972) was able to locate a few responsive female P. divisa LeConte and record the flash dialogue between male and female; Zorn and Carlson (1978) and Carlson et al. (1982) reported that they could obtain responsive virgin females of P. versicolour Fabricius and P. lucicrescens Barber by rearing large field-collected larvae. They recorded female responses to various simulated male flashes but did not report the actual male and female flash exchange. All of these authors were operating with the hypothesis that the response interval was the critical parameter in courtship communication because that is what had been shown to be important in Photinus fireflies (Lloyd 1966). However, Vencl et al. (1994) pointed out that there was more variability in this parameter than would be expected for such a critical parameter.

Most field-collected females seem to be mated and unresponsive to males of the same species (Zorn and Carlson 1978). Rearing may be the only way to obtain virgin female Photuris fireflies that are responsive to males of the same species. Several workers have reared field-collected Photuris larvae to produce adults (McLlean et al. 1972, Buschman 1984). However, field-collected larvae are likely to include larvae of several species (Buschman 1984), in addition, most Photuris adults cannot be identified to species based on morphological characteristics (Barber 1951, Lloyd 1969), so the species identity remains in question (although some authors give species names with complete confidence). I think it is best to know the species identity of the mother Photuris before the larvae are cultured.

The firefly Photuris versicolour quadriflugens Barber [1] (hereafter referred to as P. quadrifulgens) is an early season firefly (Fig. 1E) that occurs throughout Eastern North America (Forrest and Eubanks 1995, Buschman 2017). Male P. quadrifulgens produce two- to five-pulse flash patterns as they fly over pastures, hayfields, meadows, and up into trees around the perimeter of these meadows and pastures. Males also produce flickers as they fly over pastures, hayfields, and meadows. Forrest and Eubanks (1995) reported details of the male flash pattern in Mississippi, and they documented significant variation in the number of flashes in the male flash pattern. This species was chosen for study because it is the only Photuris firefly active in the early part of the season and this allowed us to identify female P. quadrifulgens fireflies with a high degree of confidence. In addition, larvae were reared from another female Photuris and the offspring of this female yielded good behavioural data. This female will be identified as Photuris tremulans Barber, because her male offspring produced single flashes at 2.7-sec intervals and because this is the most likely continuous single-flashing species known to occur at this time of year in Eastern Tennessee. The development of the larvae and the mating communication of these two Photuris firefly species will be described. These observations were also verified by building a firefly simulator to do a preliminary test of the flash communication of these fireflies in the field. 

Methods

Twelve P. quadrifulgens females were collected by watching for periodic flashing in the vegetation (flashes at ca. 1-sec intervals) and other unusual flashing in the vegetation (Buschman 2017). These females were collected from a 1.2-ha horse pasture in northwest Knox County, Tennessee, and from a 12-ha hayfield in Jefferson County, Tennessee (see Buschman & Faust 2014 2015 for more details). One P. tremulans female was collected in the short vegetation under trees on the shore of “Eslinger Swamp” (12 kilometers south of the McBee Island site). The females were housed in transparent plastic containers of various sizes (350 to 1850 ml; Fig. 1A). The containers were covered with transparent plastic wrap or later with an opaque plastic lid with two small holes. The containers included some moist native soil (mounded on one side of the container 2 cm deep). The soil was watered with an eyedropper once or twice a week. A piece of apple was provided to give them moisture/nourishment. The apple was placed on a toothpick and anchored in the soil to prevent it from rolling around and injuring the insects.

The females laid their eggs in the soil, and the eggs were allowed to hatch in the containers where they were laid. Some 30 days later, when hatching was imminent, a piece of paper towel was placed on the bottom of the container to absorb moisture and prevent larvae from getting stuck in condensation (a serious threat for first instars), and a fresh piece of apple was added to provide initial nourishment as the larvae hatched. When the first instars could be seen in the container, a small section of earthworm (from the local fish bait shop) was added for food. Feeding and watering continued once a week as they developed. The earthworm was cut up into 20 to 30 pieces and placed on small paper squares (1.0–1.5 cm2) that could be handled with forceps (Fig. 1D). This also made cleanup easier one or two days later. The small larvae were reared in the containers where they hatched because they were difficult to handle safely at this stage. When larvae reached the third and fourth instars, they started to nip at each other at feeding time, so they were transferred individually to ½-pint (237 ml) glass canning jars where they could be reared separately (Fig. 1A). The canning jars contained 1 heaping tablespoon (ca. 15 ml) of soil mounded on one side so that the other half of the floor of the jar was not covered with soil. This allowed the food to be placed on the glass floor, and leftover food could be cleaned up more easily. The soil provided environmental moisture and a place for the larvae to hide and/or build igloos for moulting or pupating. The instar stage was judged visually by observing the size difference when they came to feed. Larger larvae were weighed weekly on a Gemini-20 Portable Milligram Scale (American Weigh Scales, Inc., 3285 Saturn Court, Norcross, GA) that was sensitive to ca. 0.001 g (1mg) with weights over 10 mg.


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FIGURE 1 - A. The plastic container used to hold adult fireflies (left) and the smaller container for rearing individual fireflies (right). B. Second-instar P. tremulans larvae on a 1-cm square paper near food. C. Twelve fourth-instar P. quadrifulgens larvae on the soil inside a rearing container. D. Feeding time with earthworm pieces lined up on 1-cm square papers ready to be added to rearing containers. E. Reared adult P. quadrifulgens on an apple section in the rearing container. Note the black elytra with no vitae (the tan diagonal line is often found on Photuris elytra). Photos by L.L. Buschman.

The larvae were maintained in two homemade light-tight chambers with controlled photoperiods. A fan circulated the air from the room through the two chambers to keep the temperatures equalized, and a space heater was used to maintain a temperature of 21°C (+ 2°C). Because my objective was to study courtship communication, it was important that the larvae pupate in unison so I would have both males and females at the same time. Based on Buschman (1984), I knew that Photuris larvae were sensitive to photoperiod and they would pupate only under long-day conditions. Therefore, the larvae were reared in a photoperiod of 8 h light and 16 h dark so they could grow and develop into large larvae without pupating. The time for dusk was shifted to 1 pm to make it easier to make the night time observations when the adults emerged.

When most of the larvae were deemed ready to pupate (>50 mg), they were moved to the chamber with a long-day photoperiod, 16 h light and 8 h dark. Because more than 50 P. quadrifulgens large larvae were available for the experiment, they were divided into two groups. Group I included 27 of the oldest P. quadrifulgens larvae from Group I females (Buschman 2017) and were moved to the long-day conditions on 26 November. Group II included 25 P. quadrifulgens larvae from Group II females (Buschman 2017) together with larvae from the one P. tremulans female. Both Group II and P. tremulans larvae were moved to the long-day conditions on 17 February. Weekly feeding and watering continued until they built deep pupation igloos. Then larvae were checked more often in an effort to identify when pupation and eclosion occurred, but this was difficult because they were hidden deep in the soil. As adults emerged from pupal chambers, a small piece of apple was added to the rearing container for nourishment and moisture. Females were transferred to larger plastic containers as described earlier.

Observations of flash behavior were made in a dark room in which a large screened cage (1.8 x 1.8 x 1.8 m, Nasco #SB40675M with added floor) had been erected. Females in the larger plastic containers were arranged in order inside the cage so flash activity could be monitored individually by position. The males were either held in separate containers like the females or released into the screen cage just before darkness. Observations could then be made with knowledge of where the males and females were in the cage (at least those that were confined to their containers). Observations were recorded by voice recorder. At times females were also released into the large screen cage and allowed to mate with the males. Because the flash dialogue was found to be too rapid to describe and measure with the naked eye, some flash dialogues were recorded using a digital camera (Panasonic DMC-ZS7, Panasonic Corporation of North America, One Panasonic Way, Secaucus, NJ07094) in motion picture mode (ca. 30 frames per sec). The video recordings were converted to digital frames with a time stamp on each frame. Each frame represented 33.3 milliseconds. I could then step through the frames and record the beginning and end of each flash and calculate the length of flashes and the intervals between flash events. Flashes emitted facing the camera were recorded better than flashes emitted facing other directions. This added significant variability, particularly to the measurements of flash duration.

At the end of observations each day (usually 1 to 2 h), the fireflies in the screen cage were captured and returned to their containers to prevent them from desiccating. Observations eventually had to be terminated when males were accidentally left in the cage too long (ca. 4 h) and they died from desiccation. Fortunately, I had been able to record the critical male female flash dialogues in spite of the dry conditions. The females were in the containers where conditions were more favourable.

I will be using the flash pattern terminology of Lloyd (1966) and Lewis and Cratsley (2008), where the “flash pattern” is the unit of flashing that is separated from other such units by a longer interval. In P. quadrifulgens, there were two to five flashes in the flash pattern. The “flash interval” is the time between two consecutive flash patterns measured from the first flash in consecutive patterns. The “flash duration” is the length of the individual flash (pulse), and the “interflash interval” is the time from the beginning of one flash to the beginning of the next flash (within the flash pattern). The “response interval” in this report will be the time between the beginnings of the first male advertising flash to the first female response flash. This differs from some other definitions for response interval where the measurement is from the beginning of the last male flash to the beginning of the first female flash. I prefer the beginning of the first male advertising flash because the female P. quadrifulgens usually responded before the male advertising flash pattern was complete. Most measurements would be negative numbers if the response interval measurements were made from the last male flash.

In the laboratory, both male and female Photuris began flashing soon after the lights went out. It was not clear which of these flashes were advertising flashes, but it was clear that females would respond to some flashes and not others; therefore, the flashes associated with male and female flash dialogue were analysed separately from other flashes. Male flashes produced during the flash dialogue were similar to field advertising flashes, so these flashes were considered male “advertising flashes.” Female flashes associated with the flash dialogue were considered female “response flashes.” The parameters of these dialogue flashes were then compared with those of other recorded flashes to see if they had dialogue flash characteristics. Flashes that did not match the parameters of the dialogue flashes were then considered “other flashes,” or “periodic flashes” since they appeared to have their own rhythm. When males free in the cage were flashing on a female container, some interpretation was required to determine which flashes were male advertising flashes and which flashes were female response flashes (based on the parameters identified for dialogue flashes). Most of the recorded periodic flashes were probably produced by females, because the camera was focused on the female container. Male periodic flashes may have been present, but they could not be identified as male flashes when they were on the female container.

A portable “Firefly Simulator” (FFS) was designed and built by the KU Instrumentation Design Laboratory to test the validity of the laboratory observations. This unit was used to simulate the female flashes in the field to see if males could be attracted to simulated female flashes based on laboratory observations. The FFS was a handheld unit (3 x 6 x 12 cm) using a microcontroller that stored two flash patterns that could be played back to a light-emitting diode (LED) when the corresponding button was pressed. The flash pattern was designed with a custom software program (KU Instrumentation Design Laboratory) that ran on a PC computer.  The design software uses six parameters to control the number, length, and character of the flash trains. Flash patterns were downloaded to the FFS from the PC via the USB port. The output of the FFS microcontroller was fed to the LED as a flash-width modulated to provide intensity control and then low-pass filtered to smooth the micro-waves and create square waves with rise and fall times on the order of 4 milliseconds (ms). The LED was mounted on the end of a 1.8-m fishing pole (Lloyd, unpublished report) and connected with a 2-m cable to the FFS, which was mounted on the handle at the base of the pole. This allowed the LED to be held away from the body to avoid interfering with the approaching fireflies. The pole also allowed the observer a better view of firefly activity around the LED. Two interchangeable standard size T1-3/4 LEDs with diffused lenses and a beam divergence of 60° were used, one with a peak at 565 nm (yellowish-green) and one with peak at 585 nm (yellowish-orange).

Follow-up field observations were made 25–29 May 2013 at McBee Island, Tennessee, ca. half a mile from where some of the 2012 observations were made. The firefly simulator was programmed to present the P. quadrifulgens female response flashes to flying males as five 110-ms flashes at 190-ms intervals. The firefly simulator was programmed to present the P. tremulans female response flashes to flying males as three 240-ms flashes at 1230-ms intervals. These flash patterns were presented to flying males that were producing pulsed or flicker flash patterns. For P. quadrifulgens, the response flashes were presented anytime during the male flash pattern; presenting a more specific response time would require more practice. For P. tremulans, the response flashes were presented whenever the male was flashing; I had no way to mimic flash timing as precisely as females do. The firefly simulator was not available during the laboratory observations, so it could not be used to test female responses to other firefly flash patterns.

Results and Discussion

Photuris quadrifulgens

Species identity
The identity of the females recorded as P. quadrifulgens is as secure as any Photuris identity can be. They were collected early in the season when there were no other Photuris in the habitat. In addition, the male offspring produced typical P. quadrifulgens flash patterns. P. quadrifulgens occur early in the season, 3 May to 1 June or 626-1131 mGDDF (349-629 mGDDC) (Buschman 2017).

Rearing Observations
Ten of the 12 field-collected P. quadrifulgens females produced first-instar larvae (83%). Eggs were difficult to see in the soil except when they were laid next to the wall of the container. The eggs hatched after about 30 days, but the exact dates of oviposition and hatching were unknown. There were a total of 107 P. quadrifulgens first instars from 10 females. They were reared at a temperature of 21°C (+ 2°C).

I examined the possibility that females collected in the early part of the season (20–26 April; Group I) had more larvae than females collected in the later part of the season (30 April–2 May; Group II) and found that Group I females produced 10.4 larvae/female (range = 8–12, n = 5) and Group II females produced 10.8 larvae/female (range= 8–15, n = 7), so no clear trend was evident. I examined the possibility that the number of larvae produced might be related to the number of fireflies the females consumed (Buschman 2017). Females that ate zero or one firefly (n = 6) produced 9.8 larvae/female, and females that ate two or three fireflies (n = 4) produced 10.4 larvae/female, so no clear trend was evident. I also examined the possibility that the number of larvae produced might be related to the number of days the female lived. Females living 8–16 days (n = 4) produced 10.3 larvae/female, and females living 17–22 days (n = 6) produced 10.5 larvae/female; again, no clear trend was evident.

The newly hatched Photuris first instars were vulnerable to getting caught in surface tension of condensation in rearing containers. After the first instars took their first meal, they seemed more robust, and their chances of survival seemed to improve. Many of the small larvae molted after each meal during the first two or three weeks. First and second instars (Fig. 1B) seemed to hide in cracks and crevasses. Third and fourth instars (Fig. 1C) started building shelters and igloos. After the third instar, the interval between molts became longer than one week. Some larvae did not come out from hiding to feed at every feeding; they apparently were waiting for a molt. Third and fourth instars were more aggressive toward each other around the food—nipping at each other. At this point, they were large enough to handle safely with a soft forceps, so they were transferred to individual rearing ½-pt. jars (237 ml) (Fig. 1A).

The P. quadrifulgens larvae continued to grow steadily, and by the end of November, 30 and 29 weeks after maternal female collection, many of the larvae appeared to be ready to pupate. The larvae were divided into two groups. At the end of November, Group I larvae averaged 64 mg (range 33–99 mg). These larvae were transferred to the rearing chamber with a long-day photoperiod (16 h of light, 8 h dark), where they continued to grow and pupated in February. Seven males (Fig. 1E) emerged as adults 88 days later (range = 81–94 days) and weighed 86 mg (range = 61–131 mg) at pupation. Eleven females emerged as adults 99 days later (range = 80–125 days) and weighed 94 mg (range = 62–127 mg) at pupation. Four of these larvae died as large larvae (three from a fungus infection and one of unknown causes), and another four died during pupation (unknown cause). One larva was still alive when observations terminated.

At the end of November, Group II P. quadrifulgens larvae averaged 39 mg (range = 7–92 mg). These larvae remained in the short-day photoperiod chamber (8 h of light, 16 h of dark), where they continued to grow; in February, they averaged 57 mm (range = 24–90 mg). On February 17, these larvae were transferred to the long-day photoperiod rearing chamber (16 h of light, 8 h dark), where they continued to grow to pupation in April. Three males emerged as adults 37 days later (range = 36–40 days) and weighed 75 mg (range = 68–83 mg) at pupation. Four females emerged as adults 45 days later (range = 37–54 days) and weighed 93 mg (range = 78–114 mg) at pupation. One larva from Group II broke through the short-day photoperiod regime and pupated before the larvae were transferred to the long-day photoperiod. One of the larvae died as a large larva of unknown causes, and another five died during pupation of unknown causes. Ten larvae were still alive when observations terminated.

The three P. quadrifulgens larvae that died of a fungus infection were the offspring of one female and had been reared together as small larvae. The infection may have spread in the original container. A male held with that female had died covered with fungus. The fungus was identified as Metarhizium spp. All the larvae from this female died, probably from the same fungus. A total of four of the 12 female rearing containers had fireflies that died covered with fungus, two males and two females. All four of the containers had first instars, but two of these containers produced no adults. One chamber produced three females, and the other chamber produced one male, two dead pupae, and three live larvae. During this project, the handling utensils were conscientiously disinfected when moving from chamber to chamber, and the infection seemed to be contained within the original containers.

Flash Behaviour
In the field, flying male P. quadrifulgens produced two to five pulsed flash patterns as also described by Forrest and Eubanks (1995) and Buschman (2017). In the laboratory, both males and females began their flashing activity by producing single periodic flashes soon after the lights went out as they began to move around in their containers. After several minutes, males began producing occasional “advertising flashes,” which were two or three brighter flashes with a distinctive timing. During the next 30 to 60 min, they continued to produce occasional advertising flash patterns. Females would answer some of these advertising flashes, but would ignore the repeated single periodic flashes.

P. quadrifulgens males released in the screened cage produced many periodic flashes, but occasionally they would produce the brighter advertising flashes. Males tried to fly/hover while producing advertising flashes, but the flights were short, and most males were lucky to complete one flash pattern before they flew into the screen wall. On one occasion, a hovering male received a response from a female. This male then lit up with what appeared to be a continuous glow with various intensity modulations. This male descended to the female (the modulations in the glow may have contained a flash dialogue with the female). This happened only once and was not captured on video. This glow was similar to the glows and modulations associated with Photuris landing which Lloyd (1968) thought functioned in illumination during landing.

P. quadrifulgens females responded to male advertising flashes with a series of fast flashes that usually started during the male’s third flash, but sometimes they began as early as during the first male flash or as late as 0.5 sec after the third (and last) male flash (Fig. 2). The flash dialogue was so fast that much of it could not be timed with the naked eye. When a female responded to a male advertising flash, there seemed to be an increase in the number of advertising flash patterns in the cage.

 


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FIGURE 2 -Photuris quadrifulgens flash dialogue between two males and a female. The first male starts his advertising flash pattern of three flashes (sometimes up to five flashes). The second male joins the first male flashing in synchrony during the second flash. The female responds with three to five very fast flashes that start during the third flash of the first male. The stars indicate the starts of nine female responses. (Temperature ca. 21°C).

There were a total of 16 male P. quadrifulgens advertising flash patterns recorded for Group I males and there were 12% two-pulsed, 63% three-pulsed, 19% four-pulsed, and 6% five-pulsed flash patterns. There were a total of 11 advertising flash patterns recorded for Group II males and they were all two pulsed flash patterns. The flash duration for male advertising flashes averaged 124 ms, and the flash interval averaged 708 ms (Table 1A, Fig. 3A). Both the flash duration and the flash intervals for male advertising flashes were significantly shorter than those for periodic flashes, but significantly longer than for female response flashes (Table 1A). In the analysis of the first through fifth flashes in the male advertising flash pattern, the first flash was significantly shorter in duration than the other flashes, but the flash intervals did not differ significantly (Table 1B). The distribution of flash intervals for male flashes is shown in Fig. 3A.


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TABLE 1 -P. quadrifulgens flash durations and flash intervals for males and females in flash dialogue, advertising and response flashes, or periodic flashes (not in flash dialogue). Flashes are produced by ca. five males and five females (Temperature ca. 21°C). Means in the same column followed by different letters were significantly different (P = 0.05).


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FIGURE 3 -Photuris quadrifulgens male and female flashes: red for male “advertising” and female “response” flashes produced during flash dialogue and black for “periodic” flashes that are not produced during flash dialogue. Flashes are produced by ca. five males and five females. (Temperature ca. 21oC).

The P. quadrifulgens female response flash was a series of fast flashes that usually started during the third male flash, but this varied (Fig. 2). The 18 female response flash patterns recorded on video were 6% two-pulsed, 28% three-pulsed, 22% four-pulsed, 17% five-pulsed, 6% six-pulsed, 6% seven-pulsed, 12% eight-pulsed, and 6% nine-pulsed patterns. The flash intervals ranged from 0.1 to 0.4 sec, but they clustered between 0.1 and 0.3 sec (Fig. 3B). The intervals at 0.4 sec appeared to be multiples of the 0.2-sec interval (with a missed flash), so the mean was calculated using the intervals between 1.0 and 0.3 sec. The flash duration for female response flashes averaged 103 ms, and the flash intervals averaged 179 ms (Table 1A). Both the flash duration and the flash intervals were significantly shorter than periodic flashes and male advertising flashes. In the analysis of the first through ninth flash in the female response flash, neither the flash duration nor the flash interval differed significantly (Table 1C).

P. quadrifulgens periodic flashes (non-dialogue flashes) that were recorded on video had intervals of 0.2 to 2.1 sec, but most of them clustered between 0.8 and 1.4 sec (Fig. 3B). The interval at 0.2 sec appeared to be an outlier (another firefly?), and the intervals at ca. 2.0 sec appeared to be multiple intervals (with missing flashes), so the intervals between 0.8 and 1.4 sec were used to calculate the mean interval. The duration of periodic flashes averaged 189 ms and the interval averaged 1101ms (Table 1A). These flashes were significantly longer than male advertising flashes and female response flashes. Most of the recorded periodic flashes were probably produced by females, because the camera was focused on the female container.

Female Biology
A total of six P. quadrifulgens females were observed in the dark room on a daily basis when there were active males present. They were observed for 8 to 25 days (they were different ages). All six females produced periodic flashes throughout their lives; however, these flashes were most common during the first two weeks (based on unstructured observations).

Virgin P. quadrifulgens females produced response flashes in response to male advertising flashes, but not to periodic flashes. They began responding on day one or two after eclosing and the responses were most frequent during the first two or three days. The frequency of response seemed to decline after two or three days based on unstructured observations. One female that had been removed from its earthen cell prematurely did not flash for the first two days but then was active like the other females. The fireflies apparently need several days to complete development in the earthen cell before they emerge and become active flashers. Males were added to three of the virgin female cages. Mating was not observed, but two of the females stopped responding to male advertising flashes two to four days later. They seemed to require one or two nights and flash exchanges with the male in the container before they mated with them. Larvae were found in two of the containers and eggs were seen in the third container. The other three virgin females were released into the cage with males, one at a time. All three exchanged flashes with males and appeared to mate, staying together 22, 15, and 35 min. The male remained dorsal to the female until they separated (Phase I, Lewis and Wang 1991). The first two females did not respond to male advertising flashes after mating, and larvae were found in their containers. The third female may not have mated successfully, because she continued to respond to male advertising flashes without pause. She was released into the cage a second time and had flash exchanges with several males, but she refused to accept approaching males. No larvae were found in her container. One of the five mated females was recorded as responding to a male flash, but that happened only once, so it may have been a recording error. 

Nine P. quadrifulgens flash exchanges were recorded on video and included both male and female flashes (video #1, Supplemental information). The female response interval averaged 1152 ms (n = 9, SD = 609, range = 101–2069 ms) (Fig. 2). The extreme variation in the female response interval suggests that the response interval must not be the critical component in the courtship flash communication system. The female response flashes included two to nine flashes that began just after the first male flash to after the third male flash. Most of the female responses started during the second or third male flash.

It was interesting to note that on two occasions, a second P. quadrifulgens male synchronized his advertising flashes with those of the first male (Fig. 2). In both cases, the second male joined the flashing during the second of three flashes and flashed in unison for the two flashes. On the first occasion, the two males flashed in perfect synchrony (frame to frame), and on the second occasion, the second male was one frame behind the first male on both flashes. Lloyd (1981b) noted that male Photuris competitors sometimes flashed in synchrony with the second flash of their opponent’s advertising flash. Such synchrony may enable the male to identify a female response to the other male (Lloyd 1983). He also suggests that the second male may be able to get the female to turn her light toward him instead of toward the first male. Copeland and Moiseff (2004) analysed the “intermittent synchrony” in P. frontalis when they occurred in high populations and males approached each other.

Field response of males to the simulated female flashes
In the field, the simulated P. quadrifulgens female flashes presented with the Firefly Simulator were effective at attracting males. A total of 28 P. quadrifulgens males advertising with flashes were attracted to within 30 cm of the LED, three landed on the LED when it was held in the air above the ground, and 13 males were attracted from the tree tops, up to 12 m above. When the LED was placed on a 0.6 x 0.6 m board on the ground, many males landed and crawled to the LED. In addition, a total of 13 males advertising with flicker flashes were attracted to within 30 cm, and one landed on the LED when it was held in the air above the ground. The flicker flashers switched to pulsed flashes after they landed and were climbing to the LED. One evening, 10 of 12 pulsing males were attracted from within 3 m, and four of five flickering males were attracted. It did not seem to matter when during the male flash train the simulated female flash train started. P. quadrifulgens males did not respond to P. tremulans simulated response flashes.

Photuris tremulans

Species identity
The identity of the Photuris female collected at Eslinger Swamp is more complicated, as it is with most field-collected Photuris females. There are few usable morphological characteristics that can be used to identify Photuris fireflies. However, her male offspring produced single flashes at 2.7-sec intervals; therefore, I will be referring to these fireflies as P. tremulans because this is the most likely continuous single-flashing species known to occur at this time of year in Eastern Tennessee.

Rearing Observations
The female P. tremulans lived 22 days in captivity. During this time she ate two fireflies and produced 14 first-instar larvae. They were reared at a temperature of 21°C (+ 2°C). They averaged 33.6 mg in November and 78.3 mg in April just before they reached the pupal stage. Three female larvae averaged 85.7 mg, and 4 males averaged 72.8 mg at pupation. Seven adults emerged within six days, and five were available for the behavioural observations, two males and three females. They were observed for five to nine days.

Flash Behaviour
In the field, L.F Faust (personal communication) reports that flying P. tremulans males produce continuous strings of single short flashes and longer flicker flashes. The pulsed flashes were 0.2 sec long and 2.7 to 3.2 sec apart at 19oC. Flicker flashes averaged 0.7 sec long and 5.0 to 5.4 sec apart at 17 to 19°C. P. tremulans occur two to three weeks later in the season than P. quadrifulgens, 947 to 1612 mGDDF (late May through June).

In the laboratory, both male and female P. tremulans began flashing activity by producing single periodic flashes soon after the bright lights went out. They also flashed while the room was lit with dim lights whereas P. quadrifulgens was not active in such conditions. Two males were released initially into the screen cage, and they both crawled to a female’s container when she began flashing. They remained on this container until she stopped flashing and only then did they move away. They returned to the container when she resumed flashing. Courtship communication was obviously occurring, but I could not understand the signals. There did not seem to be a flash-answer sequence and I did not know which flashes were male and which were female. The next evening, the males were confined in separate containers arranged so I know which were males and which were females. Now it was clear that the male was flashing continuously at ca. 3-sec intervals and the female would join the male with continuous flashing at a significantly faster rate (Figs. 4-6).

Male P. tremulans advertising flash duration averaged 117 ms and the flash interval averaged 2693 ms (ca. 18°C) (Table 2A, Figs. 4-6) (see video #2, Supplemental Information). This flash duration was significantly shorter than that of female response flashes, but significantly longer than male and female periodic flashes (Table 2A). The flash intervals were also significantly longer than female response flashes and the non-dialogue periodic flashes. Analysis of the advertising flashes of the three individual males indicated that the flash durations and the flash intervals were significantly different (Table 2B). Analysis of advertising flashes also indicated there were significant differences between early and late recordings on the same day (Table 2F). The differences between the individual males could be associated with the insect motivational state, which seemed to decrease during the evening and as the insects aged. The distribution of flash intervals for the male advertising flashes was quite tight, and they were distinct from female flashes and most periodic flashes (Table 2A, Figs. 7 A-B).

The female P. tremulans response flash was a series of flashes that started during the male flash pattern and continued as a duet over many male flashes (Figs. 4-6). The females seemed to flash 2 to 2.5 times for each male flash. The flash intervals ranged from 0.6 to 3.2 sec, but they clustered between 0.6 and 1.7 sec (Figs. 7A & B), so the mean was calculated  using data from these flashes. The flash duration for female response flashes averaged 176 ms, and the flash intervals averaged 1168 ms (Table 2A). The flash duration was significantly and nearly twice as long as male advertising flashes as well as male and female periodic flashes (Table 2A). The flash intervals were significantly shorter than male advertising flash intervals, but similar to male and female periodic flashes (Table 2A). The individual females had similar flash durations (overall), but the flash duration for female #3 was significantly shorter than the other females (Table 2C). The flash duration of a highly motivated female (facing the camera) appears to average 217 to 220 ms (Table 2C). Analysis of response flashes early and late indicates there were significant differences for early and late recording on the same day (Table 2G). These differences and the differences between the individual females appeared to be associated with differences in motivational state of the female. The motivational state seemed to decrease during the evening and as they grew older. The distribution of flash intervals for the female response flashes was not very tight, and these flashes could not be distinguished from male or female periodic flashes (Table 2A, Figs. 7 A-B); however, the female flash duration was much longer (although this was difficult to see with the naked eye).


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TABLE 2 -Photuris tremulans flash durations and flash intervals for males and females in flash dialogue, advertising and response flashes, or periodic flashes (not in flash dialogue). Flashes are produced by two males and three females (Temperature ca. 21oC). Means in the same column followed by different letters were significantly different (P = 0.05).

In a more detailed analysis of mating communication in P. tremulans, female #1 seemed to respond with two flashes spaced between the two male flashes (Fig. 4), but female #3 seemed to respond with 5 flashes spaced between three male flashes, with the third flash overlapping the second male flash (Figs. 5 and 6). It was, therefore, not clear if the female was responding to each male flash individually (in a duet) or if she was responding with a set number of flashes that overlapped several male flashes (a flash-answer response). Some gaps in the female response train seemed to suggest the female was resetting the timing of her flashes. On three occasions, the female continued to flash after the male had stopped flashing, one is documented in Fig 4, five are shown in Fig 5, and four in Fig 6. This suggests that the female may be programed to flash a set number of flashes that extended beyond the next male flash, with the number of flashes probably based on the female motivational state; however, it also looks like the female can adjust the timing of her flashes to fit in a duet with the male flashes.


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FIGURE 4 -Photuris tremulans flash dialogue between male #1 and female #1 over a 40-sec period. The female flashes twice for each male flash. The last flash on the first and second lines repeats as the first flash on the second and third lines.


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FIGURE 5 -Photuris tremulans flash dialogue between male #2 and female #3 over a 41-sec period. The female flashes two and a half times for each male flash. The last flash on the first and second lines repeats as the first flash on the second and third lines.


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FIGURE 6 - Photuris tremulans flash dialogue between male #2 and female #3 over a 38-sec period. The female flashes two or three times for each male flash. The last flash on the first and second lines repeats as the first flash on the second and third lines.

 

The female P. tremulans response flashes were analysed two ways to gain some insight into how the female might be timing her flashes, first as though she was responding to each male flash with two or three flashes, and second as though the flashes were a set series of flashes (up to six flashes for this analysis). In the first analysis, the flash series started at each male flash, so only two or three flashes were in each series. In the second analysis, the series started after a gap in the female flash series (e.g., when only one flash followed a male flash). The intervals were expected to be uniform within a programed flash pattern.

In the first analysis, the response intervals for P. tremulans females were rather uniform because the intervals between the two female flashes were similar (not significantly different; Table 3B), although the interval between the male flash and the first female flash was significantly shorter than the intervals between two female flashes. The intervals for two females were similar (Table 3C), however, during the later trials, the intervals for female #1 differed from those of females #2 and 3 (Table 3D). In the second analysis, response intervals were not uniform because the third and fourth intervals were significantly shorter than the second and fifth intervals (Table 3A). Again, the interval between the male flash and the first female flash was significantly shorter. These results suggest the female may be programed to flash up to a dozen times, but she adjusts the timing of the flashes with each male flash. The difference between females could be associated with age and/or motivational state.


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TABLE 3 - Photuris tremulans flash intervals for females in flash dialogue analyzed either as a series of six flashes or as a series of three flashes for females #1, #2 and #3. Flashes are produced by two males and three females (Temperature ca. 21oC). Means in the same column followed by different letters were significantly different (P = 0.05).

P. tremulans male and female “non-dialogue flashes” or “periodic flashes” were recorded on video and had intervals of 0.1 to 3.2 sec, but most of them clustered between 0.6 and 1.6 sec (Fig. 7A & B), and these were used to calculate the mean interval. The duration of periodic flashes averaged 80 ms, and the average interval averaged 1.2 sec (Table 2A). The flash durations were significantly shorter than male advertising flashes and female response flashes. The differences between individual male and/or female periodic flashes were small (Table 2D & E). From day to day there were also small differences in periodic flashes, particularly for female periodic flashes (Table 2H & I). Both males and females produced periodic flashes throughout their lives; however, these flashes were most common during the first several days after emergence.


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FIGURE 7 - Photuris tremulans male and female flashes: red for male“advertising” and female “response” flashes produced during flash dialogue and black for “periodic” flashes that are not produced during flash dialogue. Flashes are produced by two males and three females. (Temperature ca. 21oC).

 

P. tremulans females began responding to male advertising flashes on day one or two after emerging as adults. The responses seemed more intense for the first two or three days, and the frequency of response and the duration of the response flash seemed to decline after that (based on unstructured observations). Females seemed to respond to advertising flashes, but not to periodic flashes. The interval between male flashes was the main difference between advertising and periodic flashes. The advertising flashes seemed to be brighter than the periodic flashes, but this parameter was not quantified. This parameter is subject to the position of the insect when the flash occurs. The female also would be expected to have difficulty judging the brightness of the male flash, so this parameter is thought to be less important for the female.

Males were added to the female containers on day 5 for females #2 and #3 and on day 9 for female #1. Females #1 and #2 engaged in flash exchanges with the males in their containers, and mating soon took place. The males were left in the jars overnight. Mating lasted 20 and 30 min, and the male remained dorsal to the female until they separated (Phase I, Lewis and Wang 1991). Female 3 did not mate and seemed to avoid the male in her container. Female #3 may have mated overnight with the male in her cage. The next night, there was flashing by a male and female #3, but analysis of video of this flashing indicated that it was not a mating flash dialogue but rather spontaneous female flashing, because the female did not seem to time her flashes relative to the male flashes. In addition, her flashes were of short duration like the periodic flashes. These three females were observed only 1 day after mating. They did not produce response flashes. These containers dried up accidentally, so I was not able to record whether eggs or larvae were produced by these females.

On most evenings, two P. tremulans males were active in the room. On one occasion when there were no female response flashes, both males produced advertising flashes, but  when one male was in a flash dialogue with one or more females, the other male would produce only periodic flashes that were random relative to the male and female flash exchanges. The couple in the flash dialogue seemed to ignore the male periodic flashes, even though they were difficult (for the observer) to distinguish from the female flashes except for the fact that they were random and had no relationship to the ongoing male female flash exchange.

Field response of males to the simulated female flashes
A total of 8 P. tremulans males advertising with flashes were attracted to within 30 cm, and two of them landed on the LED when it was held in the air above the ground. In addition another total of five flickering males were attracted to within 30 cm, and one landed on the LED. These males switched to pulsing flashes after receiving the simulated female flashes. The P. tremulans males did not seem as eager to land near the LED as were the P. quadrifulgens males. I did not record the total number of males tested, but the response was probably less than 50%. This may be because I was not able to perform the duet correctly. The males were probably attracted by the long female-like flashes, but when the duet did not occur, many flew away. The numbers of P. tremulans reported here are smaller because there was less time to test this firefly. The colour of the LED did not seem to make a difference for either species, so the yellow-orange LED was used most often. P. tremulans males did not respond to P. quadrifulgens simulated response flashes.

General Discussion

Rearing observations
A number of laboratories have successfully reared large field-collected Photuris larvae, and some were able to produce virgin females for behavioural studies (McLean et al. 1972, Zorn et al. 1978, Buschman 1984). There are also three reports that small numbers of Photuris adults were reared from eggs laid by Photuris females (WP Jones in McDermott 1958, D. Minnick in Lloyd 1969, McLean et al. 1972). The larvae reared in this study represent the first time significant numbers of Photuris that have been reared from eggs to produce virgin females and males of the same species for use in behavioural studies.

The number of first instars produced by Photuris females in this study (8–15) was low—compared to numbers recovered from other fireflies. Photuris first instars are larger than first instars of Photinus or Pyractomena (no measurements) so there are usually fewer of them. These field collected females may have been older females that had already laid much of their egg complement. But more likely, many firefly females refuse to lay their eggs and die filed with eggs—sometimes expelling a mass of eggs at death (personal observation). I was fortunate to get fertile eggs from these females—10 of 12 females laying eggs was unusual.

Life cycle
The time fireflies need to complete the life cycle is discussed in the literature. Some observers have suggested that two years are required (Williams 1917, Hess 1920, McLean et al 1972), whereas others have suggested that they could complete the life cycle within the same growing season (Buschman 1987). This will likely depend on geographic region and availability of food. In northern climates like New England and Canada, larvae may take two or three years to develop because the growing season is shorter. In southern climates like Florida, some individuals in the population may be able to complete the life cycle in less than one year (Buschman 1984, 1987), but it is doubtful that the whole cohort will develop in unison on a regular basis, either within a season or over several seasons. The larvae appear to develop facultatively; they pupate when they are large enough and the climatic conditions and photoperiod are right. Some of the larvae reared in this study could conceivably have completed development by the following spring in East Tennessee (one-year life cycle).

Photuris flash behaviour
Analysis of flash behaviour of Photuris fireflies must start with understanding which flashes are associated with mating behavior and which are not. In the field, most male flashes are “advertising flashes” produced as males hover/fly in the habitat (personal observations, Lloyd 1998). These flashes can be characteristic for each species and are used by researchers to identify firefly species. The advertising flashes can be single flashes, a series of flashes in a “flash pattern,” or a more complex flash pattern with flashes, flickers, and/or changes in flash intensity.

Identifying advertising flashes in the laboratory is more difficult, because most flashes do not resemble the advertising flashes seen in the field. In P. quadrifulgens, there are many single flashes, with only occasional flashes that look like advertising flashes — a group of two to six flashes that seem brighter than the other single flashes and are spaced ca. 0.7 sec apart (ca 17°C) as opposed to most flashes that were ca. 1 sec apart. When a responsive virgin female was in the cage she would respond to some of the flashes.  Therefore, I was able to identify male advertising flashes as flashes produced during male-female flash dialogue.

The flashes of P. tremulans were even more difficult to identify because all male flashes were single flashes. Again, when a responsive virgin female was in the cage, she would respond to some flashes, but not others. When I analysed the dialogue flashes, I found that the female was responding to flashes spaced 2.7 sec apart (ca. 17°C), whereas the other flashes at ca. 1 sec were ignored. Therefore, I identified male flashes that were spaced ca. 2.7 sec apart as male advertising flashes. These flashes seemed brighter than the 1-sec flashes.

There are several interesting things that need to be said about the flash characteristics of the two Photuris fireflies. In P. quadrifulgens, the main difference was the flash frequency, the female response flashes were very fast (179 ms apart) while the male advertising flashes were 0.7 sec apart. The periodic flashes were 1.1 sec apart. The flash durations were fairly similar, 0.1-0.2 sec, and I would use caution in placing too much weight on small differences in duration, because flashes facing the camera could be twice as long as similar flashes facing away from the camera (only the brightest parts of the flash are recording). In general, the male and female dialogue flashes were snappy, while the periodic flashes seemed to be “lazy flashes” (slower more gradual on and off slopes).

In P. tremulans, the main difference between male and female dialogue flashes was the flash interval, the female response flashes were 1.1 sec apart while the male advertising flashes were 2.7 sec apart. However, the interval for the female response flashes was the same as that for periodic flashes, 1.1 sec apart. How do males discriminate between periodic flashes and female flashes? In this case, there seemed to be a significant difference in flash duration, female response flashes averaged 176 ms while periodic flashes averaged 80 ms. In addition, female flashes facing the camera were well over 200 ms in duration (Table 2C). However, this makes them similar to P. quadrifulgens periodic flashes, 1.1 sec apart and 189 ms. The field trials with the FFS suggest the males are attracted to 0.2 sec flashes at 1.1 sec intervals, but they probably expect the female to join him in the flash duet which the FFS was not able to do. The periodic flashes were short (80 ms), probably reflecting dimmer flashes.

In P. quadrifulgens the number of flashes per flash pattern was variable; 2 to 5 flashes per pattern. This was similar to the situation observed in the field, 2 to 6 flashes per pattern (Forrest & Eubank 1995; Buschman 2017). The only thing that was unusual was that the Group II males produced only two flashes per flash pattern. I have no explanation for this observation. Double flashes were not common in the field. The number of flashes did not seem to be important in itself, although the smaller number would seem to reduce the number of intervals that could be timed by the female to identify the male advertising flash.

Flicker flashes
In the field, flicker flashes were observed in both P. quadrifulgens and P. tremulans, but flicker flashes were not observed in the laboratory. They seem to be facultative—produced under special conditions. In the field males of both species switched from flicker flashes to pulsed flashes when they were presented simulated female response flashes. Lloyd (1969) also reported that flickering Photuris males typically switched to flashes when presented simulated female responses. The function of flicker flashes remains unclear, but these results would suggest it must not have anything to do with courtship communication. They could be aposematic—signals to keep predators away (discussed further in Buschman 2017).

Periodic flashes
These “other” or “non-advertising” single spontaneous flashes were analysed to determine their characteristics. These flashes were produced by both males and females as they moved about on the jar, cage or vegetation. Plotting the frequency distribution showed that there was a peak at ca. 1 sec. These flashes seemed to be another type of flash. There was a peak in the frequency distribution so they seemed to have a degree of periodicity. Therefore they are being called “periodic flashes.”

Periodic flashes also occur in the field, but detailed field observations still need to be made. These flashes are usually ignored by researchers because there is little species information in them. These flashes are usually produced by fireflies that are grounded (on the vegetation). Female Photuris can be collected by watching for periodic flashes in the vegetation. Periodic flashes also can be seen in flying fireflies, but these flights are not the hovering flights that are usually seen with advertising flashes. They are higher (often tree canopy level) and appear to be straight-line (more directional) and faster, as though they were flying from one location to another. I recently observed a male Photuris "Short Crescendo" flying through the tree canopy producing periodic flashes. Then he slowed down and descended to the undergrowth and joined other males producing crescendo advertising flashes while hovering/flying slowly in the undergrowth. High-flying fireflies doing periodic flashes are often thought to be females, but I find that many are males. Periodic flashes were seen in both P. quadrifulgens and P. tremulans, and the flashes were ca. 1 sec apart in both species. In the laboratory, periodic flashes were more common than advertising flashes. In fact, the evening activity seemed to start with periodic flashes. Sometimes one male would produce advertising flashes while another produced periodic flashes. Females answered the advertising flashes and not the periodic flashes.

What I am calling “periodic” flashes may be equivalent to a number of flashes identified by other authors: “twinkling flashes” (Carlson et al. 1982), “walking flashes” (Lloyd 1975), “labyrinth flashes,” “shaking substrate flashes,” “ground walking flashes,” “twig walking flashes,” ”semi-cruising flashes” (Lloyd 1998), or “warm-up” flashes (L. Faust personal communication). Additional field observations will be needed to sort out how many of these flash types need to be recognized. The periodic flashes are difficult to associate with a particular function, but they would seem to be perfect as aposematic warning signals to keep predators at bay; for example, periodic or cruising flashes may be useful in deterring bats (Lloyd 1989, 1998, Moosman et al. 2009). In fact, it might be useful to think of firefly flashing as aposematic first and as courtship communication second—as opposed to the current thinking were sexual communication is considered first.

Courtship flash communication in Photuris fireflies
The literature on the courtship communication of Photuris fireflies in the field is reviewed by Buschman (2017). Reports on laboratory observations of Photuris courtship communication are limited and have emphasized the flash-answer component of the communication: Buschman (1972) for P. divisa, Zorn and Carlson (1978), and Vencl et al. (1994) for P. versicolour, and Carlson et al. (1982) and Carlson and Copeland (1985) for P. lucicrescens. However, it is interesting to note the tremendous variation in the female response delay in each of these reports, an unexpected result for Signal System II fireflies because the female flash response delay is understood to be the critical parameter for communication in fireflies such as Photinus and Pyractomena (Lloyd 1971, 1978, 1983). If the response delay is not the critical parameter in Photuris courtship communication, there must be some other critical parameter, but none has been documented. Lloyd (1969) suggested that the critical parameter could be flash duration or flash rate. In Luciola fireflies of Europe and Asia, the female flash duration plus the response delay have been identified as critical parameters (Papi 1969 and Takatsu et al. 2012).

The flash dialogue between male and female P. quadrifulgens has been observed in the field only one time—during a whole season of working with them (Buschman 2017). In this case it happened so quickly and unexpectedly that it was difficult to reconstruct exactly what happened. The female clearly responded to the male advertising flash pattern at a short interval. The male lit up with what appeared to be a continuous glow with various intensity modulations. The male descended directly to the female (the modulations in the glow may have contained a flash dialogue with the female). This sequence was over in seconds. Reports of Photuris courtship flash communication in the field are usually limited to one or two flash dialogues over years of study (Barber 1951; Buschman 1972, 1974; Lloyd 1969, 1979, 1981a, 1983). In most of these reports, the communication was described as flashing by both male and female that increased as the male approached the female. The courtship of P. tremulans has not been observed and detailed field studies have not been conducted.

In the laboratory, the P. quadrifulgens female response was a characteristic rapid series of flashes that often started during the third male flash, but variation in this response delay was considerable. The flash dialogue was so rapid that it required frame-by-frame video analysis. The rate of these response flashes was the identifying characteristic for the male, because that is the most striking difference between the female response flash and all the other flashes produced by males or females. It is interesting that flash communication in the field was seen as an extended glow with modulations that increased as the male approached. There was also a similar observation in the laboratory. This may be the typical response for flying males when they receive a response flash. Most of the courtship communications reported in the laboratory have included a flash answer component. This may be typical for grounded males.

In P. tremulans, the flash communication was entirely different. There was no flash-answer component, and it took many flash exchanges for the observer to understand that a flash duet was occurring. Frame-by-frame video analysis showed that the female joined the male in a flash duet where he was flashing at a slow beat while she was flashing at a faster flash rate (“beat”—to use music terminology). The female appeared to adjust the timing of her response flashes to each male flash, but this will need further experimental evidence to confirm (equipment to do such tests was not available during this study).

In this report, we see that Photuris courtship flash communication is more complicated than that observed in Photinus and Pyractomena species. There appear to be two courtship communication systems. These two systems appear to explain some of the variation in reported field observations. The P. quadrifulgens system appeared to be similar to reports of courtship communication in Photuris fireflies that have male flash patterns, such as P. divisa, P. fairchildi, P. versicolour, P. lucicrescens, and Photuris “SH.”This group also includes many of the better known aggressive mimics (Lloyd 1965) such as P. fairchildi and P. versicolour. The P. tremulans system may account for reports of courtship communication in continuous single flashing Photuris such as P. congener and P. brunnipennis. The courtship here seems to include a continuous string of flashes by both male and female.These fireflies may not be common aggressive mimics, but they are probably facultative predators.  These Photuris do not have a flash-answer component in their courtship communication that can be modified for aggressive mimicry. However, Lloyd (1975) has described aggressive mimicry for P. congener where a string of continuous single flashes was able to attract prey. It should be noted that male fireflies are sometimes extremely curious and will investigate almost any flashing in the vegetation, including periodic flashing, flashing of captured fireflies in spider webs (personal observation) and continuous flashing LED lights (Lloyd 1983).

The Photuris courtship communication appears to have some similarities with the courtship communication in Luciola lucitanica in Italy (Papi 1969). The male advertising flash was a continuous series of single flashes at ca. 1-sec intervals. The female response was a series of single flashes emitted at a fixed interval after each male flash. This is similar to the dialogue in P. tremulans (I would call this a duet), except that the female flash rate (beat) is the same as the male flash rate (beat) (Papi refers to this as 1 to 1 flashing). In P. tremulans the two sexes have different flash rates (beats). In L. lucitanica, the frequency of the male flashes increased until the female started flashing after every other male flash. This change in frequency did not occur in P. tremulans.

P. tremulans, males and females were able to carry on a flash duet with the male and female flashing on different beats. This is not a true synchrony, but in the field it would be hard to tell the difference. Lloyd (1975) reported that P. congener seemed to carry on what I am calling a flash duet between male and female (based on aggressive mimic flashes that attracted males). Lloyd (1969, 1983) suggested the courtship in P. brunnipennis floridana seemed to involve synchrony. He also reported that males synchronize with an artificial blinker that was flashing at a rate similar to the male rate, and they walked rapidly toward such a light.

Responsiveness of mated females
During these observations, five mated P. quadrifulgens females did not appear to respond to male advertising flashes. There was only one recorded response to a male advertising flash, but this could have been a recording error. Unfortunately, these mated females were followed for only a few days before observations were accidently terminated. A sixth mated female responded to the male advertising flashes, but appeared to avoid approaching males even after extended flash dialogues. This female apparently died a virgin, because she produced no viable eggs or first instars. The three mated P. tremulans females also did not appear to respond to male advertising flashes, but they also were observed for only one or two days after mating. In both species, regular flashing ended when copulation began and the couple remained motionless in copula for some time (ca. 30 min) (but there can be occasional flashes during copulation). L. Faust (personal communication) observed random flashing and periodic body vibration by a Photuris male during copulation.

Virgin females did not readily mate with a male that was added to the female cage. Mating seemed to require the flash dialogue (in a larger space?). Judging by when females quit responding to male advertising flashes; it seemed to take several evenings for such couples to mate. When virgin females were released into the larger cage with males, one at a time, they could be seen to engage in the flash dialogue that quickly led to mating (mating within seconds to minutes). This contrasts with other fireflies that will readily mate when they make contact in a small container (Buschman 1984, 1987).

Field verification of these flash dialogues
The return field observations of 2013 seemed to verify key parts of the laboratory observations of Photuris flash dialogue. Future observations will need to focus on the Photuris flash duet between males and females. The flash duet appears to be important in Photuris courtship, as noted also by Lloyd (1969, 1975). Observations that I can interpret as the flash duet have been reported by several observers, but not by others. It is not clear if the flash duet is not always present, if it is not always recognized, or if it is confined to one group of Photuris. In Photuris fireflies, the fast flash duet may be more important than the flash response interval we are used to measuring in Photinus and Pyractomena courtship communication. These observations will require more sophisticated testing equipment and protocols. In addition, we need more field observations on periodic flashing and flicker flashing as well as the interaction of these flashes with predator such as bats. However, in spite of our current limitations, we now have a much better understanding of two mating protocols for Photuris fireflies and a better understanding of the parameters that need to be tested.

Acknowledgements

I thank James E. Lloyd, Phil Sloderbeck, Tim Forrest and an anonymous reviewer for useful suggestions on previous versions of this manuscript. I thank David A. Buschman for converting the video so it could be analysed frame by frame; Kenneth L. Ratzlaff, director of the Instrumentation Design Laboratory at the University of Kansas for building the “Firefly Simulator;” and Richard A. Humber, Insect Mycologist and Curator/ARSEF USDA-ARS Biological Integrated Pest Management Research Unit for identifying a fungus specimen. This is contribution no. 15-110-J from the Kansas Agricultural Experiment Station, Manhattan, Kansas. Twenty P. quadrifulgens and 15 P. tremulans involved in this research have been deposited in the KSU Museum of Entomological and Prairie Arthropod Research with the voucher number 235.

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