Nitric oxide synthase (NOS) in the Japanese fireflies Luciola lateralis and Luciola cruciata

Species-specific flash patterns in firefly species are important for the investigation of the evolution of Lampyridae. Since nitric oxide synthase (NOS) is one of the key enzymes controlling flash patterns, we determined the cDNA sequences of NOS in the Japanese fireflies Luciola lateralis and L. cruciata. The identity of the NOS sequences was very high between these 2 species. Firefly NOS also exhibited a high identity with those of other insect species, and the cofactor-binding domains were particularly well conserved. Many negatively selected sites were detected throughout the NOS sequences; however, no positive selection was detected. The phylogenetic relationship of insect NOS was different from that of the general classification system, although the lineages corresponded to the major recognized taxonomic groups.     

Tuning of photoreceptor spectral sensitivity in fireflies (Coleoptera: Lampyridae)

Sexual communication between male and female fireflies involves the visual detection of species-specific bioluminescent signals. Firefly species vary spectrally in both their emitted light and in the sensitivity of the eye, depending on the time when each is active. Tuning of spectral sensitivity in three firefly species that occupy different photic niches was investigated using light and electron microscopy, microspectrophotometry, and intracellular recording to characterize the location and spectral absorption of the screening pigments that filter incoming light, the visual pigments that receive this filtered light, and the visual spectral sensitivity. Twilight-active species had similar pink screening pigments, but the visual pigment of Photinus pyralis peaked near 545 nm, while that of P. scintillans had a λ_max near 557 nm. The night-active Photuris versicolor had a yellow screening pigment that was uniquely localized, while its visual pigment was similar to that of P. pyralis. These results show that both screening and visual pigments vary among species. Modeling of spectral tuning indicates that the combination of screening and visual pigments found in the retina of each species provides the best possible match of sensitivity to bioluminescent emission. This combination also produced model sensitivity spectra that closely resemble sensitivities measured either with electroretinographic or intracellular techniques. Vision in both species of Photinus appears to be evolutionarily tuned for maximum discrimination of conspecific signals from spectrally broader backgrounds. Ph. versicolor, on the other hand, appears to have a visual system that offers a compromise between maximum sensitivity to, and maximum discrimination of, their signals. Accepted: 29 September 1999     

Spectral sensitivity of the compound eyes in two day-active fireflies (Coleoptera: Lampyridae: Lucidota)

Summary The electroretinographic visual spectral sensitivity functions in day-active fireflies Lucidota luteicollis and Lucidota atra show a broad green sensitivity and a shoulder in the near-ultraviolet region of the spectrum (Figs. 1, 2) as is commonly found among day-active insects. The nomogram for P530 visual pigment matches the spectral sensitivity curves in the green. The adult L. luteicollis retains its larval bioluminescent light organ which has a peak emission at 562 nm. The _max of the ERG spectral sensitivity does not match the bioluminescent peak (Fig. 1B) as it does in twilight- and dark-active fireflies. Some relevant behavioural observations with respect to mating are presented.     

Gene diversity and geographic differentiation in mitochondrial DNA of the Genji firefly, Luciola cruciata (Coleoptera: Lampyridae)

The Genji firefly, Luciola cruciata, is divided into two ecological types, the fast-flash and slow-flash types, on the basis of the interflash interval of mate-seeking males. To evaluate the evolutionary origin of the two types, 62 populations were examined by restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase (CO) II gene. As a result, 19 haplotypes were detected, and their distributions were indigenous to local areas. Phylogenetic trees constructed from sequence comparison of the haplotypes revealed three major clades (I, II, and III). The boundary of haplotypes between clades I and II is approximately concordant with the geological structure of the Japanese Islands, which is a great rupture zone called the Fossa Magna, and the distribution of haplotypes in clades III and I-II corresponds to the Kyushu and Honshu-Shikoku Islands, respectively. The results suggest a vicariant scenario in which current L. cruciata diversity would have arisen from phylogenetic separations subsequent to the formation process of the Japanese Islands based on the molecular clock. The CO II gene trees also suggested that the fast-flash type should be considered an ancestral form, while the slow-flash type would be a derived one. The divergence time between the slow- and the fast-flash types is estimated to be about 4.6 to 2.0 mya (the Pliocene epoch).     

Modification of spectral sensitivities by screening pigments in the compound eyes of twilight-active fireflies (Coleoptera: Lampyridae)

1. ERG S(lambda) were determined in dark-adapted intact preparations of 6 North American firefly species (Photinus collustrans, marginellus, pyralis, macdermotti, scintillans and Bicellonycha wickershamorum) which restrict their flashing activity to twilight hours. The curves possess narrow (1/2 bandwidth = 50-60 nm) peaks in the yellow (560-580 nm) and a shoulder in the violet (370-420 nm), with a marked attenuation (1.4-2.2 log units) of sensitivity in the green (480-530 nm) region of the spectrum (Fig. 1). Two additional species (Photuris potomaca and frontalis) which initiate flashing at twilight and continue on late into the night (twi-night) possess broad sensitivity maxima around 560 nm (Fig. 3). 2. Selective adaptation experiments isolated near-UV and yellow in P. scintillans (Fig. 2). In the dorsal frontal region of the compound eyes in P. frontalis, high sensitivity existed only in the short wavelength region (near-UV and blue) with a maximum in the blue (lambda max 435 nm) (Fig. 4). 3. The in situ MSP absorption spectrum of the screening pigments was determined in preparations of firefly retina. a) Two kinds of dark brown granules were found in the clear zone region. These granules absorb all across the spectrum with a gradual increase in optical density in the shorter wavelength region in P. pyralis (Fig. 5). b) Besides dark granules, pink-to-red colored screening pigments were present in the vicinity of the rhabdoms. The absorption spectra of these pigments determined in five species were narrow (1/2 bandwidth = 50-80 nm) with species-specific differences in their peak absorption in the green at 525 nm, 510 nm, 512 nm and 517 nm in P. scintillans, macdermotti, collustrans and pyralis, respectively (Fig. 6). A similar pigment was found in P. marginellus with a lambda max at 512 nm (Fig. 7). In all cases, transmission increased both at long and short wavelengths, but more sharply in the long wavelength region (Figs. 6 and 7). Hence each twilight-restricted species has its own unique colored screening pigment. A yellow pigment whose absorption spectrum differed from those found in genus Photinus was found in twi-night active Photuris potomaca (lambda max 461 nm) and night-active P. versicolor (lambda max 456 nm). The transmission of the Photuris pigment increased sharply only in the long wave-length region (Fig. 8).(ABSTRACT TRUNCATED AT 400 WORDS)     

Phyletic aspects of the distribution of 3-hydroxyretinal in the class insecta

Summary The distribution of 3-hydroxyretinal (R₃), a recently discovered retinoid used as the visual pigment chromophore in some insects, was investigated in the class Insecta using HPLC technology. We studied 138 species in 24 orders, sampling from a wide range of taxonomic groups as well as varied habitats. In addition to groups already known to have R₃, we find this retinoid in Hemiptera (suborder Heteroptera), Plecoptera, Megaloptera, and Hymenoptera. We also find retinal (R₁) in Hemiptera (suborder Homoptera), Mecoptera, and Trichoptera, groups previously thought to have only R₃. The pattern of R₃ occurrence indicates that this retinoid cannot be considered a phylogenetic marker, having a scattered distribution in the class Insecta as well as within some orders of insects. Several environmental factors that might influence the selection of chromophore have been considered, but none correlates with its distribution. The evolutionary reasons for the pattern of occurrence of R₃ therefore remain unknown.     

The Habitat Requirement of the Genji-Firefly Luciola cruciata (Coleoptera : Lampyridae), a Representative Endemic Species of Japanese Rural Landscapes

Raising public interest in nature through conserving species of high social interest is crucial in achieving effective conservation of biodiversity. In Japan, the Genji-firefly Luciola cruciata (Coleoptera Lampyridae) in biodiversity rich agricultural landscapes called the satoyama has always attracted exceptional public interest. This study provides rare information on environmental factors associated with the abundance of the Genji-firefly. Stepwise backward multiple regression revealed that firefly abundance increased with increasing pH, DO and prey abundance while decreasing with water depth and the proportion of artificially modified ditch length. These factors are thought to be influential mainly to the larval and pre-pupal periods of the firefly. The implications of the results for the conservation of the Genji-firefly are discussed, with reference to the relationship between Genji-firefly conservation and extensive biodiversity conservation in the satoyama.     

The ecological impact of an introduced population on a native population in the firefly Luciola cruciata (Coleoptera: Lampyridae)

In Japan, Tatsuno Town has been famous for many Luciola cruciata fireflies emerging every summer at least since the 1920s. However, in the 1960s, L. cruciata fireflies were intentionally introduced from the Lake Biwa area into Matsuo-kyo, the most famous habitat of fireflies in that town. In this study, I examined ambient temperatures and flash rates of L. cruciata at four sites including Matsuo-kyo in the Tatsuno area and two sites in the Lake Biwa area. The linear regression of flash rates on temperatures indicated that the Matsuo-kyo population was distinct from the other three populations native to the Tatsuno area, but similar to the two populations native to the Lake Biwa area in terms of flash rates. These results were also supported by a recent molecular biological study, suggesting that the introduced fireflies had a strong ecological impact on the native ones at Matsuo-kyo. The present study emphasizes that we should not transport and release L. cruciata fireflies without careful consideration.     

Vicariance of Pyrocoelia fireflies (Coleoptera: Lampyridae) in the Ryukyu islands,Japan

We performed molecular phylogenetic analyses based on the mitochondrial COI gene (687 bp) and the nuclear 28S rRNA gene (715 bp) and reconstructed phylogenetic trees of the Pyrocoelia fireflies in the Ryukyu Islands and eastern Asia. Age calibration was done using a robust geological constraint: the Okinawa trough and associated straits began to rift at 1.55 Ma, isolating the Ryukyu Islands from the Chinese continent, Japanese islands, Taiwan island and some of the islands from each other. We suggest that the physical isolation of these islands began to generate the allopatric speciation within these islands, so the timing of this isolation was assigned to an appropriate node. The topology is completely concordant among phylogenetic trees reconstructed using MEGA (maximum‐likelihood), raxmlGUI (maximum‐likelihood) and BEAST (Bayesian inference; including combined analysis of COI and 28S rRNA genes). Two lineages are recognized, related to their emergence time; spring to summer, and autumn. In each lineage, vicariance is inferred to have begun at 1.55 Ma from our phylogenetic and geological analyses. In lineage 1, P. oshimana (Amami), P. matsumurai (Okinawa), P. discicollis (W. Japan), P. fumosa (E. Japan) and P. abdominalis (Yaeyama) were differentiated. In lineage 2, P. rufa (Tsushima and Korea), P. miyako (Miyako‐jima), P. atripennis (Ishigaki‐jima) and P. praetexta (Taiwan and HongKong) were differentiated. Pyrocoelia analis (Taiwan and China) emerges throughout the year except for winter, and constitutes another lineage. We suggest that Pyrocoelia fireflies differentiated at 2 Ma to generate these three lineages. The base substitution rate for the COI gene is estimated as 4.48% Myr^–1 and that for the 28S rRNA gene is 0.394%  Myr^–1, and these rates were used in a combined BEAty analysis in BEAST.     

Phylogeographic Analysis of the Firefly, Luciola lateralis, in Japan and Korea Based on Mitochondrial Cytochrome Oxidase II Gene Sequences (Coleoptera: Lampyridae)

Luciola lateralis is widely distributed throughout the Korean Peninsula, northeast China, Sakhalin, and Japan. Two ecological types are recognized in Japan based on flash and hatching time characteristics. The mitochondrial cytochrome oxidase II gene was surveyed by restriction fragment length polymorphism analysis for Japan (46 populations) and Korea (two populations). Eleven haplotypes were detected. Gene trees revealed that haplotypes between Japan and Korea are much more differentiated in nucleotide sequences (8.1%) than those within Japan (0.3-1.4%) and Korea (0.7%). Haplotypes between Honshu and Hokkaido are not separated as clades, and the two ecological types cannot be segregated from each other phylogenetically. We suggest that the Japanese populations of this species may have dispersed within one million years ago and that ecological differences may be the result of physiological adaptation to cold climates.     

Morphology of the alimentary and reproductive organs of Luciola discicollis Cast. (Coleoptera: Lampyridae)

The morphology of the alimentary and reproductive organs of L. discicollis (Coleoptera: Lampyridae) was studied in detail. The alimentary canal possesses a pouch-like proventriculus. It is suggested that this structure was once functional in ancestral Lampyridae. The details of the reproductive organs are given and it appears that they might be of value in determining the inter-relationships of the families included in the superfamily Cantharoidea. Male and female specimens of L. discicollis have been found to exhibit sexual dimorphism particularly marked in respect of the gut and the size of the eyes.     

Bioluminescence emissions of the firefly <Emphasis Type="Italic">Luciola praeusta</Emphasis> Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae)

We recorded the in vivo emission and time-resolved spectra of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae). The emission spectrum shows that the full width at half maximum (FWHM) value for this particular species is 55 nm, which is significantly narrower than the in vivo half-widths reported till now. The time-resolved spectrum reveals that a flash of about 100 ms duration is, in fact, composed of a number of microsecond pulses. This suggests that the speed of the enzyme-catalysed chemiluminescence reaction in the firefly for the emission of light is much faster than was previously believed. A version of this article has already appeared in an archive Nature Precedings; see     

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly,Luciola lateralis

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.     

Flash behavior of femalePhoturis versicolor fireflies (Coleoptera: Lampyridae) in simulated courtship and predatory dialogues

FemalePhoturis versicolor fireflies attempt to capture males by responding to heterospecific flash patterns. A mating-dependent switch occurs which affects response timing and frequency of female flashes. We examined the switch using females of known age, mating status, and flash experience to assess how accurate mimicry is, what factors influence it, and what mechanism produces it. Presentations of simulated male flash patterns before and after mating revealed elements of an entrainment mechanism controlling female responsiveness. Unmated females preferentially answered conspecific patterns with variable latencies, averaging 1 s. Mating induced changes in both response frequency and response latency: Females answered heterospecific patterns more frequently, and latencies elicited by conspecific patterns shifted away from the unmated range. Heterogeneity in mean and variance of response latency among individuals indicates that females do not share a discrete reply to a given pattern. Little correspondence exists between latencies of sympatric species andP. versicolor females, suggesting that the flash response mechanism produces entriainment to any rhythmic pattern, not a one-to-one matching between prey and predator latencies. Different selective scenarios underlie strict mimicry versus entrainment mimicry.     

栉角雪萤的荧光酶基因结构及其系统发育分析

短角窗萤属是萤科第四大属,但未见有该属物种荧光酶基因的研究报道。通过对总基因组的PCR扩增,对该属的栉角雪萤荧光酶基因进行了测序分析。基因序列长1958bp。与已知荧光酶基因进行同源性比较后推断,栉角雪萤的荧光酶基因由7个外显子和6个内含子组成,编码547个氨基酸残基;由推导的氨基酸序列进行同源性比较后发现,栉角雪萤的荧光酶基因与同一亚科中Lampyrini族和Cratomorphini族分别具有93—94％和92％相似性,而与北美萤火虫Photinus pyralis（Photinini族）的相似性较低（83％）。系统发育分析进一步表明栉角雪萤与Pyrocoelia、Lampyris、Cratomorphus和Photinus同属于萤亚科,且与前3个属的亲缘关系较近。这在一定程度上与形态（Branham＆Wenzel,2003）及线粒体DNA（Lietal,2006）系统发育分析所得结果一致。     

The origin of photic behavior and the evolution of sexual communication in fireflies (Coleoptera: Lampyridae)

Through a phylogenetic analysis using adult morphological characters, we show that the origin of bioluminescence in cantharoid beetles appears to predate the origin of the family Lampyridae. The ability to produce and emit photic signals was first gained by larvae and appears to function as an aposematic warning display; it was subsequently gained in adults and is used as a sexual signal. Our analysis also suggests that while pheromonal sexual signals are used basally in the family, they are used in conjunction with and then subsequently replaced by photic signals in some lampyrid lineages. Both photic signals and the photic organs used to produce them have become greatly elaborated in the fireflies that no longer employ pheromonal sexual signals. In addition, the ability to produce a flashed sexual signal appears to have arisen at least three times in the family Lampyridae. Convergent evolution is also evident in a number of adult male photic organ morphologies. Further, we recommend that individual signal system components be compared rather than overall signal system complexity. The use of this strategy may allow one to recognize and better interpret adaptive correlations despite convergence or loss. We demonstrate that phylogenetic analysis is a powerful tool even for rapidly evolving traits.     

Correlated evolution of female neoteny and flightlessness with male spermatophore production in fireflies (Coleoptera: Lampyridae)

The beetle family Lampyridae (fireflies) encompasses ～100 genera worldwide with considerable diversity in life histories and signaling modes. Some lampyrid males use reproductive accessory glands to produce spermatophores, which have been shown to increase female lifetime fecundity. Sexual dimorphism in the form of neotenic and flightless females is also common in this family. A major goal of this study was to test a hypothesized link between female flight ability and male spermatophore production. We examined macroevolutionary patterns to test for correlated evolution among different levels of female neoteny (and associated loss of flight ability), male accessory gland number (and associated spermatophore production), and sexual signaling mode. Trait reconstruction on a molecular phylogeny indicated that flying females and spermatophores were ancestral traits and that female neoteny increased monotonically and led to flightlessness within multiple lineages. In addition, male spermatophore production was lost multiple times. Our evolutionary trait analysis revealed significant correlations between increased female neoteny and male accessory gland number, as well as between flightlessness and spermatophore loss. In addition, female flightlessness was positively correlated with the use of glows as female sexual signal. Transition probability analysis supported an evolutionary sequence of female flightlessness evolving first, followed by loss of male spermatophores. These results contribute to understanding how spermatophores have evolved and how this important class of seminal nuptial gifts is linked to other traits, providing new insights into sexual selection and life-history evolution.     

Variation in flash speed of Japanese firefly,Luciola cruciata (Coleoptera: Lampyridae), identifies distinct southern “quick‐flash” population on Goto Islands,Japan

Luciola cruciata, a bioluminescent firefly endemic to Japan, communicates with species‐specific flashing patterns. Previous studies reported that flying males possess three distinct flashing frequencies: slow (4 s), intermediate (3 s) and fast (2 s). Our study focused on geographical variations in flashing patterns in the northwestern part of the Kyushu region of southern Japan (including its isolated islands) and their relationship to the mitochondrial COII region of firefly DNA. We found that “quick‐flash (1 s)” fireflies occur in the Goto Islands, at the western tip of Kyushu, and that, although they possess a unique haplotype, they do not differ much genetically from “fast‐flash” fireflies inhabiting mainland Kyushu. In contrast, although fireflies inhabiting the Tsushima and Iki Islands also possess a unique haplotype, their flashing patterns were the same (fast‐flash) as fireflies inhabiting mainland Kyushu. We determined that flash patterns of L. cruciata in Japan should be classified according to four flashing patterns: slow, intermediate, fast and quick.     

条背萤幼虫不同发育阶段呼吸系统构造的观察和比较

通过显微解剖,结合透射电镜和扫描电镜,观察比较了条背萤Luciola substriata幼虫两个不同发育阶段呼吸系统的差异。结果表明：1～2龄幼虫的呼吸系统中只有气管无气囊,3～6龄幼虫的呼吸系统中气管和气囊并存。1～2龄幼虫的尾气门和3～6龄幼虫的腹部侧气门及尾气门结构没有差异。透射电镜观察提示条背萤1～2龄幼虫体壁上的毛状物为气管鳃。